U.S. patent number 11,268,117 [Application Number 16/308,803] was granted by the patent office on 2022-03-08 for methods and compositions for nucleic acid amplification.
This patent grant is currently assigned to Life Technologies Corporation. The grantee listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Theo Nikiforov, Abraham Rosenbaum, Hua Yu.
United States Patent |
11,268,117 |
Yu , et al. |
March 8, 2022 |
Methods and compositions for nucleic acid amplification
Abstract
In some embodiments, the disclosure relates generally to
methods, as well as related compositions and kits for
recombinase-mediated nucleic acid amplification, such as
recombinase-polymerase amplification (RPA), of a nucleic acid
template using at least one blocked primer that contains a 5'
domain, at least one nucleotide that is cleavable by an RNase H
enzyme, a 3' domain, wherein the primer is not extendable by a
polymerase, and wherein the 3' domain has a length of 7-100
nucleotides, for example 10-30 nucleotides. These methods and the
use of a blocked primer reduce or eliminate non-specific
amplification products, such as primer dimers, which are generated
in RPA reactions.
Inventors: |
Yu; Hua (Guilford, CT),
Nikiforov; Theo (Carlsbad, CA), Rosenbaum; Abraham
(Waterbury, CT) |
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
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Assignee: |
Life Technologies Corporation
(Carlsbad, CA)
|
Family
ID: |
1000006160153 |
Appl.
No.: |
16/308,803 |
Filed: |
June 9, 2017 |
PCT
Filed: |
June 09, 2017 |
PCT No.: |
PCT/US2017/036842 |
371(c)(1),(2),(4) Date: |
December 10, 2018 |
PCT
Pub. No.: |
WO2017/214561 |
PCT
Pub. Date: |
December 14, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20190284597 A1 |
Sep 19, 2019 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62348402 |
Jun 10, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6853 (20130101); C12Q 1/6844 (20130101); C12P
19/34 (20130101); C12Q 1/6844 (20130101); C12Q
2521/101 (20130101); C12Q 2521/327 (20130101); C12Q
2521/507 (20130101); C12Q 2525/121 (20130101); C12Q
2525/186 (20130101) |
Current International
Class: |
C12P
19/34 (20060101); C12Q 1/6853 (20180101); C12Q
1/6844 (20180101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO-03072805 |
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Sep 2003 |
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WO |
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WO-2009135093 |
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Nov 2009 |
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WO |
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WO-2009150467 |
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Dec 2009 |
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WO |
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WO-2011060014 |
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May 2011 |
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WO |
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WO-2012083189 |
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Jun 2012 |
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WO |
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WO-2012135053 |
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Oct 2012 |
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WO |
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WO-2013023176 |
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Feb 2013 |
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WO |
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WO-2013123238 |
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Aug 2013 |
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WO |
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WO-2013142364 |
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Sep 2013 |
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WO |
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WO-2014110528 |
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Jul 2014 |
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WO |
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WO-2015195949 |
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Dec 2015 |
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WO |
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Other References
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Pourmand, N et al., "Direct electrical detection of DNA synthesis",
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Sequencing", IEEE ISCAS 2002 Proceedings, Circuits and Systems,
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XP002501560, pp. 1115-1121. cited by applicant .
Dobosy J.R et al., "RNase H-Dependent PCR (rhPCR): Improved
Specificity and Single Nucleotide Polymorphism Detection Using
Blocked Cleavable Primers", BMC Biotechnology, 2011, vol. 11, No.
80, pp. 1-18, URL: https://doi.org/10.1186/1472-6750-11-80. cited
by applicant .
Dobosy J.R et al., "RNase H-Dependent PCR (rhPCR): Improved
Specificity and Single Nucleotide Polymorphism Detection Using
Blocked Cleavable Primers (Supplementary Material)", BMC
Biotechnology, 2011, vol. 11, No. 80, pp. 1-13, URL:
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PCT/US2017/036842, dated Aug. 29, 2017, 28 pages. cited by
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Hoser, M.J., et al. (2014) PLoS One 9(11): e112656.
doi:10.1371/journal.pone.0112656 "Strand Invasion Based
Amplification (SIBA.RTM.): A Novel Isothermal DNA Amplification
Technology Demonstrating High Specificity and Sensitivity for a
Single Molecule of Target Analyte", Supplemental Information. cited
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Kanaya et al., "Expression, Purification, and Characterization of a
Recombinant Ribonuclease H from Thermus thermophilus HB8," The
Journal of Biological Chemistry, May 15, 1992 (May 15, 1992), vol.
267, pp. 10184-10192. cited by applicant.
|
Primary Examiner: Wilder; Cynthia B
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Application filed under 35
U.S.C. .sctn. 371 of International Application No.
PCT/US2017/036842, filed on Jun. 9, 2017, which claims the benefit
of priority under 35 U.S.C. .sctn. 119 to U.S. provisional
application No. 62/348,402, filed Jun. 10, 2016; the disclosures of
all the aforementioned applications are incorporated by reference
in their entireties.
Claims
What is claimed is:
1. A method for amplifying a double-stranded nucleic acid template,
comprising: a) forming a reaction mixture by combining the
double-stranded nucleic acid template, a polymerase, a recombinase,
a forward primer, a reverse primer, and an endonuclease, wherein
the forward primer binds to a forward primer binding site on a
first strand of the nucleic acid template and the reverse primer
binds to a reverse primer binding site on a second strand of the
nucleic acid template, wherein the forward primer or both of the
forward primer and the reverse primer is not extendable by the
polymerase, and wherein the non-extendable primer comprises a 5'
domain and a 3' domain separated by a cleavable moiety that is
cleavable by the endonuclease, wherein the 5' domain is at least 15
nucleotides in length and the 3' domain is 15 to 30 nucleotides in
length, the recombinase to bind to the forward primer or both of
the forward primer and the reverse primer; and b) incubating the
reaction mixture under substantially isothermal amplification
conditions, whereby the recombinase binds to the forward primer and
invades the double-stranded nucleic acid at the forward primer
binding site, the endonuclease cleaves the cleavable moiety of the
forward primer, and the 5' domain of the forward primer is extended
by the polymerase, thereby amplifying the nucleic acid
template.
2. The method of claim 1, wherein the forward primer and the
reverse primer are not extendable by the polymerase.
3. The method of claim 1, wherein the 5' domain is 15 to 30
nucleotides in length and/or the 3' domain is 15 to 20 nucleotides
in length.
4. The method of claim 1, wherein a 3' nucleotide of the 3' domain
of the non-extendable primer is mismatched to the forward primer
binding site.
5. The method of claim 1, wherein the cleavable moiety comprises a
ribonucleotide.
6. The method of claim 1, wherein: the forward primer is a first
universal primer, the reverse primer is a second universal primer,
and the first universal primer and the second universal primer are
not extendable by the polymerase, the reaction mixture comprises at
least two different nucleic acid templates comprising both a
forward primer binding sequence and a reverse primer binding
sequence, wherein the forward primer binding sequence is
complementary or identical to at least a portion of the first
universal primer and the reverse primer binding sequence is
complementary or identical to at least a portion of the second
universal primer, the reaction mixture is in contact with a support
having the first universal primer bound thereto, and at least two
substantially monoclonal nucleic acid populations are formed by
using the polymerase to amplify each of the at least two different
nucleic acid templates onto different sites on the solid support
within the same reaction mixture.
7. The method of claim 6, further comprising sequencing the at
least two substantially monoclonal nucleic acid populations.
8. The method of claim 1, wherein: the reaction mixture comprises
(i) a plurality of template nucleic acids, each template nucleic
acid including a first and second universal primer binding site,
(ii) a plurality of forward primers, (iii) a plurality of reverse
primers, and (iv) a plurality of nucleotides, the forward primer is
non-extendable and includes a blocking moiety at the 3' terminal
end of the primer that prevents primer extension, wherein at least
a portion of the 5' domain of the forward primer can hybridize with
the first universal primer binding site, and wherein at least a
portion of the 3' domain can hybridize with the first universal
primer binding site; the reverse primer is extendable and includes
at least a portion that hybridizes with the second universal primer
binding site, and the method comprises: amplifying the plurality of
template nucleic acids with the plurality of reverse primers to
generate a first plurality of amplification products, wherein the
reaction mixture for amplifying with the plurality of reverse
primers to generate a first plurality of amplification products
does not include the endonuclease; and amplifying the plurality of
template nucleic acids and the first plurality of amplification
products with the plurality of forward and reverse primers under an
isothermal amplification condition to generate a second plurality
of amplification products, wherein the amplifying that generates a
second plurality of amplification products is performed in the
reaction mixture that does include an endonuclease.
9. The method of claim 1, wherein the reaction mixture further
comprises a recombinase accessory protein.
10. The method of claim 9, wherein the recombinase accessory
protein is a single-stranded binding protein and/or a recombinase
loading protein.
11. The method of claim 1, wherein the amplifying is at a
temperature from 35.degree. C. to 45.degree. C.
12. The method of claim 1, wherein the incubating is for 15 to 60
minutes.
13. The method of claim 2, wherein the forward primer and the
reverse primer comprise a 5' domain and a 3' domain separated by a
cleavable segment comprising one or more nucleotides that are
cleavable by the endonuclease, wherein the 5' domain is 10 to 100
nucleotides in length and the 3' domain is 11 to 30 nucleotides in
length.
14. The method of claim 1, wherein the non-extendable primer is
immobilized to a solid support.
15. The method of claim 1, wherein the nucleic acid template is a
member of a nucleic acid library comprising a population of nucleic
acid templates each comprising a forward primer binding sequence,
and wherein the forward primer is a universal forward primer that
binds the universal forward primer binding sequence and is not
extendable by the polymerase.
16. The method of claim 15, wherein the nucleic acid templates each
comprises a reverse universal primer binding sequence and wherein
the reverse primer is a universal reverse primer that binds the
universal reverse primer binding sequence and is not extendable by
the polymerase.
17. The method of claim 1, wherein either or both of the forward
primer and the reverse primer are immobilized on a solid
support.
18. The method of claim 17, wherein the solid support is a
bead.
19. The method of claim 1, wherein the endonuclease is an RNase H
enzyme.
20. The method of claim 19, wherein the RNase H enzyme is E. coli
RNase HIT.
Description
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
This application includes an electronically submitted sequence
listing in .txt format. The .txt file contains a sequence listing
entitled "LT01140US_ST25.txt" created on Dec. 10, 2018 and is 6,000
bytes in size. The sequence listing contained in this .txt file is
part of the specification and is hereby incorporated by reference
herein in its entirety.
Throughout this application, various publications, patents, and/or
patent applications are referenced. The disclosures of the
publications, patents and/or patent applications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
BACKGROUND
Nucleic acid amplification is very useful in molecular biology and
has wide applicability in practically every aspect of biology,
therapeutics, diagnostics, forensics and research. Generally,
amplicons are generated from a starting template using one or more
primers, where the amplicons are homologous or complementary to the
template from which they were generated. Multiplexed amplification
can also streamline processes and reduce overheads. This
application relates to methods and reagents for nucleic acid
amplification and/or analysis using cleavable primers.
One example of such amplification is Recombinase Polymerase
Amplification (RPA) which is a DNA amplification process that
utilizes enzymes that hybridize oligonucleotide primers to their
complementary partners in DNA (e.g., duplex DNA) followed by
isothermal amplification. RPA offers a number of advantages over
traditional methods of DNA amplification. These advantages include
the lack of a need for any initial thermal or chemical
denaturation, the ability to operate at low constant temperatures
(e.g., isothermal conditions) without a need for absolute
temperature control, as well as the observation that complete
reactions (lacking target) can be stored in a dried condition.
These characteristics demonstrate that RPA is a uniquely powerful
tool for developing portable, accurate, and instrument-free nucleic
acid detection tests. However, use of standard primers in RPA
methods may result in nonspecific amplification product and/or
primer dimer products, which reduce the efficiency of the reaction
especially in the instance of next gen sequencing. Furthermore,
primer design constraints are a drawback of RPA.
SUMMARY
Herein are provided blocked primers (e.g., oligonucleotide primers)
containing a ribose base separating a 5' domain and a 3' domain of
the primers, optionally for use in amplification reactions,
especially isothermal reactions such as recombinase polymerase
amplification (RPA). The ribose base moiety can be cleaved by
certain endonuclease enzymes such as RNase H. The use of at least
one such blocked primer (e.g., forward and/or reverse
oligonucleotide primers), and an endonuclease that cleaves
ribobase(s) (e.g. RNase H) after primer binding to a template DNA,
reduces nonspecific amplification products providing an improved
method for amplification of nucleic acid. Specific and surprising
configurations for such primers have been identified, that provide
effective primers for reactions that involve endonuclease cleavage
of the primers, such as amplification reactions. Amplification
reactions can include but are not limited to PCR (Polymerase Chain
Reaction), HCR (Hybridization Chain Reaction), RCA (Rolling Circle
Amplification), RPA (Recombinase Polymerase Amplification), LAMP
(Loop mediated isothermal amplification), HDA (Helicase Dependent
Amplification), cluster-generation methods such as bridge
amplification (e.g. U.S. Pat. Appln. No. 2008/0009420 (Schroth et
al.)) and "template-walking" (e.g. U.S. Pat. Appln. No. 2012/083189
(Li et al.)).
Methods, reagents and products of nucleic acid amplification and/or
analysis are provided herein. In some embodiments, the present
teachings provide compositions, systems, methods, apparatuses and
kits for nucleic acid amplification.
Methods are provided for cleaving a double-stranded nucleic acid
with an endonuclease, comprising the steps of (1) forming a
reaction mixture comprising template nucleic acids (e.g., nucleic
acid templates) and primers which include a cleavable moiety (e.g.,
a ribose base), wherein the primers are at least partly
complementary to the template nucleic acid, (2) exposing the
resulting mixture to conditions suitable for hybridization between
the primers and the template nucleic acids, and (3) cleaving the
primers at the cleavable moiety with the endonuclease, where the
primers are at least partly hybridized to the template nucleic
acids. In some embodiments, the endonuclease selectively cleaves
the primers. In some embodiments, the endonuclease does not cleave
the primers at other nucleotide positions. In some embodiments, the
endonuclease cleaves a significant fraction of primers. In some
embodiments, the endonuclease cleaves the primers less efficiently
at elevated temperatures. In some embodiments, the endonuclease is
RNase H2. In some embodiments, the cleavable moiety is a ribose
base (e.g., a ribonucleotide). In some embodiments, the cleaving
step is performed at a temperature below 60.degree. C. (e.g. at
room temperature or about 20-50.degree. C., 20-30.degree. C.,
20-40.degree. C., 25-40.degree. C., 30-40.degree. C., 35-40.degree.
C., or 40-50.degree. C.). In some embodiments, the reaction mixture
is contacted with amplification reagents and/or subjected to
amplification conditions. In some embodiments, the reaction mixture
further comprises a plurality of second primers that are
reverse-complementary to the template nucleic acid, and the second
primers optionally comprise a cleavable moiety (e.g., a ribose
base), where the cleavable moiety is situated more than 5
nucleotides away from the 3' end of the oligonucleotide (e.g., at
least 7, 10, 12, 15 or 20 nucleotides).
In some embodiments, the disclosure relates to methods for cleaving
one or more blocked primers, comprising the steps of forming a
reaction mixture by combining a nucleic acid template, a forward
primer, a reverse primer, an RNase H enzyme, and optionally a
source of reactive nucleotides such as dNTPs, and optionally a
buffer, wherein the forward primer binds to a forward primer
binding site on the nucleic acid template and the reverse primer
binds to a reverse primer binding site on the nucleic acid
template, wherein one or both of the forward or reverse primers is
blocked, and wherein the blocked primer comprises a 5' domain and a
3' domain separated by at least one cleavable nucleotide (e.g., a
ribobase), wherein the 5' domain is at least 10 nucleotides in
length (e.g. 10 to 100 nucleotides in length) and the 3' domain is
at least 10 nucleotides in length (e.g. 10 to 100, 10 to 90, 10 to
80, 10 to 75, 10 to 60, or 10 to 50 nucleotides in length); and
optionally incubating the reaction mixture under substantially
isothermal, or isothermal, amplification conditions between
20.degree. C. and 50.degree. C. (e.g. 25.degree. C. and 40.degree.
C., 30.degree. C. and 40.degree. C., or 35.degree. C. and
40.degree. C.) for 10 minutes to 120 minutes, thereby amplifying
the nucleic acid template. In an embodiment the reaction mixture
optionally comprises at least one or more of: a polymerase, a
recombinase, a single-stranded binding protein, or a recombinase
loading protein.
In some embodiments, the disclosure relates to methods for cleaving
a blocked primer, comprising the steps of forming a reaction
mixture by combining a nucleic acid template having a forward
primer binding sequence and a reverse primer binding sequence, and
a blocked forward primer (i.e. non-extendable primer; i.e. forward
primer that is not extendable by a polymerase), a reverse primer
which is optionally blocked, an RNase H enzyme, and optionally a
buffer comprising a divalent cation, wherein the forward primer
binding sequence is complementary or identical to at least a
portion of the blocked forward primer and the reverse primer
binding sequence is complementary or identical to at least a
portion of the blocked reverse primer, and wherein the blocked
forward primer and the blocked reverse primer comprise a 5' domain
and a 3' domain separated by at least one cleavable nucleotide
(e.g. comprising a ribobase), wherein the 5' domain is at least 10
(e.g. 10 to 70, 10 to 60, 10 to 50, or 10 to 40) nucleotides in
length and the 3' domain is at least 10 (e.g. 10 to 70, 10 to 60,
10 to 50, 10 to 40 or 10 to 25) nucleotides in length; and
optionally incubating the reaction mixture under substantially
isothermal amplification conditions (e.g., between 20.degree. C.
and 50.degree. C. (e.g. 25.degree. C. and 40.degree. C., 30.degree.
C. and 40.degree. C., or 35.degree. C. and 40.degree. C.)) for 15
minutes to 60 minutes, thereby amplifying the nucleic acid
template. In some embodiments, the reaction mixture optionally
comprises at least one or more of: a polymerase, a recombinase, a
single-stranded binding protein, or a recombinase loading
protein.
In some embodiments, the disclosure relates to methods for nucleic
acid amplification, comprising the steps of forming a reaction
mixture by combining at least two different polynucleotide
templates comprising both a first primer binding sequence and a
second primer binding sequence, a recombinase, a recombinase
accessory protein, a polymerase, a first blocked universal primer,
a second optionally blocked universal primer, an RNase H enzyme,
and optionally dNTPs and a buffer, wherein the reaction mixture is
in contact with a support having the first blocked universal primer
bound (e.g., immobilized) thereto, wherein the first primer binding
sequence is complementary or identical to at least a portion of the
first blocked universal primer and the second primer binding
sequence is complementary or identical to at least a portion of the
second blocked universal primer, and wherein the first blocked
universal primer and the second blocked universal primer comprise a
5' domain and a 3' domain separated by a nucleotide comprising a
ribobase, w wherein the 5' domain is 10 to 70, 10 to 60, 10 to 50,
or 10 to 40 nucleotides in length and the 3' domain is 10 to 70, 10
to 60, 10 to 50, 10 to 40 or 10 to 25 nucleotides in length; and
forming at least two substantially monoclonal nucleic acid
populations by using the polymerase to amplify each of said at
least two different polynucleotide templates onto different sites
on the solid support, within the same reaction mixture of step (a)
under substantially isothermal conditions. The amplified monoclonal
nucleic acid populations may be sequenced.
In some embodiments, the blocked forward primer and the blocked
reverse primer comprise a 5' domain and a 3' domain separated by a
nucleotide comprising a ribobase, wherein the 5' domain is 10 to
100 nucleotides in length and the 3' domain is 11 to 30 nucleotides
in length. In embodiments the 5' domain is 15 to 25 nucleotides in
length or greater than 25 nucleotides. In some embodiments, the 5'
domain 15 to 50 nucleotides in length. In some embodiments, the
ribobase is rU, rG, rC or rA.
In some embodiments, the 3' domain is 14 to 25 nucleotides in
length or 15 to 25 nucleotides in length. In some embodiments, a 3'
nucleotide of the 3' domain of the forward primer is mismatched to
the forward primer binding sequence.
In some embodiments the recombinase is selected from the group
consisting of uvsX, RecA, RadA, RadB, Rad 51, a homologue thereof,
a functional analog thereof and a combination thereof. In some
embodiments, the reaction mixture comprises uvsY accessory protein
and uvsX recombinase.
In some embodiments, the RNase H enzyme is RNase HII. In some
embodiments, the RNase H enzyme is RNase HII and the incubating
temperature is between 35.degree. C. and 42.degree. C.
In some embodiments, the RNase H enzyme is present at a
concentration from 5 U to 200 U/50 .mu.L or from 10 to 90 U/50
.mu.L.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a non-limiting schematic of RPA with RNase H enzyme
cleavage methods. A standard RPA is shown in the top portion of
FIG. 1. An RPA with RNase H cleavage is shown in the lower portion
of FIG. 1. The two inverted ovals around single chain represent
recombinase bound to oligonucleotide. The open rectangles represent
single stranded binding protein. The solid diamond represents a
blocking moiety. The single vertical oval across two strands
represents RNase bound to double stranded nucleic acid. The 3/4
open circle represents a polymerase.
FIG. 2 is a non-limiting schematic of blocked primers and exemplary
primer configurations, including exemplary 5' domain and 3' domain
lengths.
FIG. 3 is a photo of a gel showing the results of blocked primer
configuration screening with V1, V2 and V3 primers by comparing DNA
template amplification using the blocked primers.
FIG. 4 is a photo of a gel showing the results of blocked primer
configuration screening with V4 and V5 primers by comparing DNA
template amplification using the blocked primers.
FIG. 5 is a photo of a gel showing the results of blocked primer
configuration screening with V4 and V5 primers by comparing DNA
template amplification using the blocked prime with longer exposure
to detection reagent.
FIG. 6 is a photo of a gel showing the results of DNA template
amplification wherein nonspecific amplification using a
non-template control reduced or eliminated using blocked primers of
the invention.
FIG. 7 is a photo of a gel showing the results of DNA template
amplification wherein nonspecific amplification is reduced or
eliminated using blocked primers of the invention.
FIG. 8A is a photo of two gels showing the results of RNase H
enzyme unit titration by analyzing DNA amplification using various
concentrations of RNase H enzyme in an RPA reaction with blocked
primers.
FIG. 8B is a photo of a gel showing the results of RNase H enzyme
unit titration by analyzing DNA amplification using various
concentrations of RNase H enzyme in an RPA reaction with blocked
primers.
FIG. 8C is a photo of a gel showing the results of DNA
amplification with a range of amplification reaction
temperatures.
FIG. 9 is a photo of a gel showing the results of DNA amplification
using two different pellet formulations of enzymes rehydrated in an
RPA reaction with blocked primers of the invention and regular
primer controls.
FIG. 10 is a list of exemplary blocked primer and adapter
sequences.
FIG. 11 is a list of exemplary primer sequences for amplification
on a solid surface.
FIG. 12A is a table showing the reaction volumes and times for an
amplification sequencing reaction with results shown in FIG.
12B.
FIG. 12B is a table that lists a comparison of DNA template
amplification on a solid support using RNase H cleavable blocked
primers of the invention followed by sequencing of the amplified
template.
FIG. 12C is a read length histogram for sequencing results using
the reaction volumes and times of FIG. 12A.
FIG. 13 is a photo of a gel showing the results of DNA
amplification using Endo IV endonuclease and an abasic cleavable
blocked primer in an RPA reaction.
FIG. 14 is a photo of a gel showing the results of DNA
amplification using APE 1 endonuclease and an abasic cleavable
blocked primer in a RPA reaction as compared to a ribobase
cleavable blocked primer and RNase HII enzyme.
FIG. 15 is a photo of a gel showing the results of DNA
amplification using the endonuclease APE 1 or Endo IV and an abasic
blocked primer in a RPA reaction as compared to a ribobase
cleavable blocked primer and RNase HII enzyme and controls.
DETAILED DESCRIPTION
In some embodiments, reducing nonspecific amplification including
reducing primer dimer formation in nucleic acid amplification
reactions. In some embodiments, the reduced non-specific
amplification can be achieved in an isothermal amplification
reaction, for example using a recombinase-mediated amplification
reaction such as RPA (recombinase-polymerase amplification). In
some embodiments, simplified primer designs can be used for such
reactions. In some embodiments, the methods, compositions, and kits
use blocked primers that comprise one or more cleavable moieties
(e.g., ribose bases) that separate a 5' domain and 3' domain of the
primer, wherein an enzyme (e.g., ribo-endonuclease, such as RNase
H), cleaves the primer at the cleavable moiety location thereby
removing the blocking moiety. The 5' domain of the primer remains
hybridized to the template nucleic acid while the 3' domain is
removed. In some embodiments, the methods, compositions and kits
identify surprising ranges for 5' and especially 3' domain
nucleotide lengths of the blocked primers. These domain lengths,
discussed below in detail, surprisingly result in efficient
amplification of the template nucleic acid and surprisingly reduce
or even eliminate primer dimer product formation and nonspecific
amplification. In some embodiments, the methods include clonal
amplification that utilize recombinase-mediated amplification and
the improved blocked primers. In some embodiments, the methods
include using especially effective concentration ranges (excess
concentration) for RNase H in such recombinase amplification
reactions using blocked primers that include a ribobase.
In some embodiments, therefore amplifying a nucleic acid template,
that includes forming a reaction mixture by combining the nucleic
acid template (e.g., a template polynucleotide), a polymerase, a
recombinase, a forward primer, a reverse primer, wherein at least
one of the forward primer or the reverse primer is a blocked
primer, dNTPs, an RNase H enzyme, and a buffer. In some
embodiments, the blocked primer is a blocked forward primer that
binds to a forward primer binding site on the nucleic acid template
and the reverse primer binds to a reverse primer binding site on
the reverse complement of the nucleic acid template. The blocked
forward primer comprises a 5' domain and a 3' domain separated by
at least one nucleotide comprising a ribobase and a blocking group
on the 3' end of the primer. The reaction mixture, in some
embodiments, is incubated under substantially isothermal
amplification conditions to amplify the nucleic acid template.
In some embodiments, methods for amplifying nucleic acid
template(s) upstream of sequencing methods. Nucleic acid templates
for these embodiments can be at least some, and typically all
members of a nucleic acid sequencing template library. In some
embodiments, the method includes forming a reaction mixture by
combining at least two different polynucleotide templates
comprising both a first primer binding sequence and a second primer
binding sequence, a recombinase, a recombinase accessory protein, a
polymerase, a first blocked universal primer attached to a support,
a second optionally blocked universal primer, dNTPs, an RNase H
enzyme, and a buffer, wherein the reaction mixture is in contact
with the support, wherein the first primer binding sequence is
complementary or identical to at least a portion of the first
blocked universal primer and the second primer binding sequence is
complementary or identical to at least a portion of the second
blocked universal primer. The polymerase, by amplifying the at
least two different polynucleotide templates, forms at least two
substantially monoclonal nucleic acid populations onto different
sites on the solid support, within the same reaction mixture of the
first step under substantially isothermal conditions. This
multi-clonal population of amplified nucleic acid template may then
be used in sequencing workflow methods, such as high throughput
sequencing methods.
In some embodiments, the methods use recombinase to denature, or
partially denature, double stranded nucleic acid templates, which
can be carried out at isothermal conditions with a polymerase, and
is referred to as recombinase-polymerase amplification (RPA) (see,
e.g., WO2003072805, hereby incorporated by reference in its
entirety). In some embodiments, the partial denaturation and/or
amplification, including any one or more steps or methods described
in the teachings herein, can be achieved using a recombinase and/or
single-stranded binding protein. Suitable recombinases include RecA
and its prokaryotic or eukaryotic homologues, or functional
fragments or variants thereof, optionally in combination with one
or more single-strand binding proteins (SSBs). In some embodiments,
the recombinase optionally binds single-stranded DNA (ssDNA) such
as the blocked primers, to form a nucleoprotein filament strand
which invades a double-stranded region of homology on a template.
See FIG. 1. This optionally creates a short hybrid and a displaced
strand bubble known as a D-loop. In FIG. 1, the free 3'-end of the
hybridized primer after cleavage by the RNase H enzyme is extended
by DNA polymerases to synthesize a new complementary strand. The
complementary strand displaces the originally-paired partner strand
of the template as it elongates. In some embodiments, the one or
more of a pair of blocked primers are contacted with one or more
recombinases before being contacted with a template which is
optionally double-stranded.
In any of the methods described herein, amplification of a template
optionally comprises contacting at least one blocked primer with a
template strand, which optionally has a region of complementarity
to at least one blocked primer. After hybridization of the blocked
primer to the template DNA, the blocked primer is cleaved at the
cleavable moiety (e.g., ribose base) location thereby liberating
the 3' domain of the blocked primer. The newly formed 3' end of the
5' domain of the primer is then extended along the template with
one or more polymerases (e.g., in the presence of dNTPs) to
generate a double stranded nucleic acid and a displaced template
strand. The amplification reaction can comprise repeated cycles of
such contacting and extending until a desired degree of
amplification is achieved, including, In some embodiments,
substantially monoclonal amplification of the template DNA.
Optionally the displaced strand of nucleic acid is amplified by a
concurrent amplification reaction. Optionally, the displaced strand
of nucleic acid is amplified by contacting it in turn with one or
more complementary blocked primers and extending the complementary
primer (after cleavage with an RNase H enzyme) by any strategy
described herein. Optionally before a blocked primer is contacted
with a template nucleic acid, it is first contacted with an
amplification enzyme (e.g. a recombinase or a polymerase) which
binds to the primer. Any amplification enzyme that has not
associated with the one or more blocked primers is optionally
removed.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits which utilize at least one
blocked RNase cleavable primer in the amplification of template
nucleic acid. The blocked primer can be the forward primer, reverse
primer or both. A forward primer and reverse primer typically form
a primer pair for amplification. The forward primer binds a forward
primer binding sequence in a forward direction on a forward strand.
The reverse primer binds to a reverse primer binding sequence in a
reverse direction on the complement strand of the forward strand.
The blocked primers can be universal primers, which can, for
example, bind to a target sequence in a gene or other sequence of
interest, bind to a sequence found in a plasmid cloning vector, or
in some embodiments, bind to universal adaptors found on or near
the ends of template nucleic acids of a nucleic acid library. In
some embodiments, one or more of the universal primers does not
contain any target (template) specific sequences. In some
embodiments, both the forward and reverse blocked primers of the
invention are universal primers, which hybridize to a universal
adapter sequence in the nucleic acid template. See Example 5. In
some embodiments, only one of the forward and reverse blocked
primers of the invention is a universal primer. Provided at least
one primer is a blocked primer, then the other primers, forward or
reverse, can be standard (non-blocked) primers. In some
embodiments, the disclosure relates generally to methods, as well
as related compositions and kits, wherein blocked primers that are
cleavable by RNase H. In some embodiments, the blocked primers
contain at least four components; a 5' domain, at least one
ribobase, which is part of a cleavable segment, a 3' domain and a
blocking moiety (See FIG. 2). Such primers can be referred to
herein, for example, as "blocked primers", "non-extendable primer"
or "blocked RNase cleavable primers." The ribobase, for example a
ribonucleotide, when the primer is hybridized to a DNA template, is
susceptible to cleavage by ribo-endonucleases, thereby separating
the 5' domain and the 3' domain. See FIG. 2. Surprisingly, primers
that included long domains, especially long 3' domains, were
effective in reducing or eliminating primer dimer product
formation. In some embodiments, the 3' domain is at least 7, 10, 12
or 14 nucleotides long, e.g. 14 to 30 nucleotides in length. In
some embodiments, the blocked primer is between 15 and 200
nucleotides long, and includes a ribobase that is more than 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the 3'
terminus of the blocked primer, referred to in the alternative
embodiment herein, as the oligonucleotide. It is noteworthy that
the Alternative Embodiments herein include oligonucleotides that
are not extendable by a polymerase (i.e. blocked oligonucleotides).
Such blocked oligonucleotides include blocked primers that are
cleavable by RNase H, as well as other oligonucleotides that are
cleavable by other enzymes, as provided in the Alternative
Embodiments section herein.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits, which use at least
oligonucleotide that is not extendable by a polymerase, such as one
blocked primer that is cleavable by RNase H. In some embodiments,
the blocked primer is a blocked forward primer. In some
embodiments, the blocked primer is a blocked reverse primer. In
some embodiments the blocked primers are complementary or identical
to the template nucleic acid. In some embodiments, the blocked
primers are universal forward and/or reverse primers, i.e.,
complementary or identical to multiple different templates that
comprise different sequences. In some embodiments, the forward or
the reverse primer is blocked. In some embodiments, both the
forward and reverse primers are blocked.
In some embodiments, the one or more blocked primers comprise a
"forward" primer and a "reverse" primer. Placing both primers and
the template in contact optionally results in a first double
stranded structure at a first portion of said first strand and a
double stranded structure at a second portion of said second
strand. Optionally, the 3' end of the forward and/or reverse primer
(after cleavage by RNase H enzyme) is extended with one or more
polymerases to generate a first and second double stranded nucleic
acid and a first and second displaced strand of nucleic acid.
Optionally, the second displaced strand is at least partially
complementary to each other and can hybridize to form a daughter
double stranded nucleic acid which can serve as double stranded
template nucleic acid in a subsequent amplification cycles.
In some embodiments, In some embodiments, a blocked primer, forward
primer and/or reverse primer, used in any of the methods provided
herein includes a 5' domain and a 3' domain separated by at least
one nucleotide comprising a ribobase. In some embodiments, the 5'
domain and 3' domain are separated by a single ribobase. In some
embodiments, the 5' domain and 3' domain are separated by
consecutive ribobases, such as two, three, four, five or more
ribobases. For example, the 5' domain and 3' domain can be
separated by between 1 and 5 consecutive ribobases. In some
embodiments, the ribobases are rU, rG, rA, or rC.
In some embodiments, the 3' domain of the blocked primers contain a
blocking moiety, which is removed after cleavage at the ribobase
location, once hybridized to the DNA template, with an RNase H
enzyme. The block, or blocking group, is a chemical moiety on the
end of the 3' primer and prevents primer extension, effectively
blocking nucleic acid amplification. Once the blocking group is
removed, the hybridized 5' domain of the primer is capable of
participating in primer extension and RPA nucleic acid
amplification. In some embodiments, the blocking group can be any
moiety that prevents or blocks primer extension. In some
embodiments, the block is a C3 spacer, a phosphate, biotin, or
amine moiety.
In some embodiments, the 5' domain of the blocked primer can be any
length, but is typically at least 10 nucleotides in length. In some
embodiments, the 5' domain is between 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90
nucleotides on the low end of the range, and 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100
nucleotides on the high end of the range. In some embodiments, the
5' domain is typically at least 15 nucleotides in length and
accordingly, in some embodiments is between 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70,
80 or 90 nucleotides on the low end of the range, and 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55,
60, 70, 80, 90, or 100 nucleotides on the high end of the range. In
some embodiments, the 5' domain can be at least 25 nucleotides in
length and accordingly, in some embodiments the 5' domain is
between 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 45, 50, 55, 60, 65, 70, 80 or 90 nucleotides on the low end of
the range, and 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 100 nucleotides on the
high end of the range. In some embodiments, the 5' domain is 30
nucleotides in length.
In some embodiments, the 5' domain can be between 10 and 60
nucleotides in length, and accordingly In some embodiments, the 5'
domain is between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 35, 40, 45, 50 or 55 nucleotides on the low end of the range,
and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,
55 or 60 nucleotides on the high end of the range. In some
embodiments, the 5' domain can be between 15 and 60 nucleotides in
length, and accordingly in some embodiments, the 5' domain is
between 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,
50 or 55 nucleotides on the low end of the range, and 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55 or 60
nucleotides on the high end of the range. In some embodiments, the
5' domain can be between 25 and 60 nucleotides in length, and
accordingly In some embodiments, the 5' domain is between 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 40, 45, 50 or 55
nucleotides on the low end of the range, and 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 40, 45, 50, 55 or 60 nucleotides on the high
end of the range. In some embodiments, the 5' domain can be between
10 and 40 nucleotides in length, and accordingly In some
embodiments, the 5' domain is between 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30 or 35 nucleotides on the low end of the
range, and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 30, 35 or 40 nucleotides on the high end of the range. In some
embodiments, the 5' domain can be between 15 and 40 nucleotides in
length, and accordingly in some embodiments, the 5' domain is
between 15, 16, 17, 18, 19, 20, 25, 30 or 35 nucleotides on the low
end of the range, and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,
35 or 40 nucleotides on the high end of the range. In some
embodiments, the 5' domain can be between 25 and 40 nucleotides in
length, and accordingly in some embodiments, the 5' domain is 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40
nucleotides in length.
In some embodiments, the 5' domain can be between 10 and 30
nucleotides in length, and accordingly in in some embodiments, the
5' domain is between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides on the low end of
the range, and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25 or 30, nucleotides on the high end of the range. In some
embodiments, the 5' domain can be between 15 and 30 nucleotides in
length, and accordingly in some embodiments, the 5' domain is
between 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or
29 nucleotides on the low end of the range, and 16, 17, 18, 19, 20,
21, 22, 23, 24, 25 or 30, nucleotides on the high end of the
range.
In some embodiments, the blocked primers comprise a 5' domain that
has a length of at least 10 nucleotides, at least 15 nucleotides,
at least 25 nucleotides or 30 or more nucleotides. In some
embodiments, the blocked primers comprise a 5' domain with a range
of nucleotide lengths from 10 to 100, 10 to 60, 10 to 50, 10 to 40,
10 to 30 or 10 to 25. In some embodiments, the blocked primers
comprise a 5' domain with a range of nucleotide lengths from 15 to
100, 15 to 60, 15 to 50, 15 to 40, 15 to 30 or 15 to 25. In some
embodiments, the blocked primers comprise a 5' domain with a range
of nucleotide lengths from 25 to 100, 25 to 60, 25 to 50, 25 to 40
or 25 to 30.
In some embodiments, the 5' domain can be between 15 and 25
nucleotides in length. See V5 primer configuration of FIG. 2, FIG.
10, Table 2 of Example 1 and corresponding FIGS. 4 and 5. In some
embodiments, the 5' domain can be 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25 nucleotides in length. In some embodiments, the 5'
domain can be 15 or 17 nucleotides in length. In some embodiments,
the 5' domain of the blocked primers can be at least 25 nucleotides
in length. See V2 primer configuration of FIG. 2, FIG. 10, Table 1
of Example 1 and corresponding FIG. 3. In some embodiments, the 5'
domain can be between 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 45, 50 or 55 nucleotides on the low end of the
range, and 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 45, 50, 55, 60, 70, 75, or 80 nucleotides on the high end of
the range. In some embodiments, the 5' domain can be between 30,
31, 32, 33 or 34 nucleotides on the low end of the range, and 31,
32, 33, 34 or 35 nucleotides on the high end of the range.
In some embodiments, the 3' domain is at least 10 nucleotides in
length, but can be 7, 8 or 9 nucleotides in length. In some
embodiments, the 3' domain is not less than 10 nucleotides in
length and in some embodiments the 3' domain is not less 6
nucleotides in length. In some embodiments, the 3' domain is
between 10 and 30 nucleotides in length, wherein the 3' domain is
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29 or 30 nucleotides in length. In some embodiments, the 3'
domain is typically at least 14 nucleotides in length. In some
embodiments, the 3' domain is between 14 and 30 nucleotides in
length, wherein the 3' domain is 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some
embodiments, the 3' domain of the blocked primers is between 15 and
30 nucleotides in length, and accordingly In some embodiments, the
3' domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29 or 30 nucleotides in length. In both the V2 and V5 primer
configuration illustrated in FIG. 2 and FIG. 10; and in the
Examples section herein, the 3' domain was at least 14 nucleotides
in length.
In some embodiments, the 3' domain is between 10 and 25 nucleotides
in length, and accordingly in some embodiments, the 3' domain is
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25
nucleotides in length. In some embodiments, the 3' domain is
between 14 and 25 nucleotides in length, and accordingly in some
embodiments, the 3' domain is 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24 or 25 nucleotides in length. In some embodiments, the 3'
domain of the blocked primers is between 15 and 25 nucleotides in
length, and accordingly in some embodiments, the 3' domain is 15,
16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In
some embodiments, the 3' domain of the blocked primers is between
14 and 20 nucleotides in length, and accordingly in some
embodiments, the 3' domain is 14, 15, 16, 17, 18, 19 or 20
nucleotides in length. In some embodiments, the 3' domain of the
blocked primers is between 15 and 20 nucleotides in length, and
accordingly in some embodiments, the 3' domain is 15, 16, 17, 18,
19 or 20 nucleotides in length.
In some embodiments, the blocked primers comprise a 5' domain and a
3' domain with a length as disclosed herein. In some embodiments,
the blocked primers comprise a 5' domain with a length between 10
and 100 nucleotides and a 3' domain with a length between 10 and 30
nucleotides, and accordingly in some embodiments, the 5' domain is
between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,
45, 50, 60, 70, 80 or 90 nucleotides on the low end of the range,
and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,
60, 70, 80, 90, or 100 nucleotides on the high end of the range and
the 3' domain is between 10 and 30 nucleotides, and accordingly in
some embodiments, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some
embodiments, the blocked primers comprise a 5' domain with a length
between 10 and 100 nucleotides and a 3' domain with a length
between 14 and 30 nucleotides, and accordingly in some embodiments,
the 5' domain is between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90 nucleotides on the low
end of the range, and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides on the high
end of the range and the 3' domain is between 14 and 30, and
accordingly in some embodiments, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some
embodiments, the blocked primers comprise a 5' domain with a length
between 10 and 100 nucleotides and a 3' domain with a length
between 15 and 30 nucleotides, and accordingly in some embodiments,
the 5' domain is between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90 nucleotides on the low
end of the range, and 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides on the high
end of the range and the 3' domain is between 15 and 30 nucleotides
in length, and accordingly in some embodiments, the 3' domain is
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 on the low
end of the range, and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29 or 30 nucleotides in length on the high end of the
range. In some embodiments, the blocked primers comprise a 5'
domain with a length of 30 nucleotides and a 3' domain with a
length of 15 nucleotides.
In some embodiments, the blocked primers comprise a 5' domain with
a length between 15 and 60 nucleotides and a 3' domain with a
length between 10 and 30 nucleotides, wherein the 5' domain is
between 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,
50 or 55 nucleotides on the low end of the range, and 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55 or 60
nucleotides on the high end of the range and the 3' domain is 10,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
nucleotides on the low end of the range, and 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length
on the high end of the range. In some embodiments, the blocked
primers comprise a 5' domain with a length between 15 and 60
nucleotides and a 3' domain with a length between 14 and 30
nucleotides, wherein the 5' domain is between 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or 55 nucleotides on the
low end of the range, and 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
30, 35, 40, 45, 50, 55 or 60 nucleotides on the high end of the
range and the 3' domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some
embodiments, the blocked primers comprise a 5' domain with a length
between 15 and 60 nucleotides and a 3' domain with a length between
15 and 30 nucleotides, wherein the 5' domain is between 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or 55
nucleotides on the low end of the range, and 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55 or 60 nucleotides on the
high end of the range and the 3' domain is 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
In some embodiments, the blocked primers comprise a 5' domain with
a length between 25 and 60 nucleotides in length and a 3' domain
with a length between 10 and 30 nucleotides, wherein the 5' domain
is between 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 40, 45,
50 or 55 nucleotides on the low end of the range, and 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55 or 60 nucleotides on the
high end of the range and the 3' domain is 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
nucleotides in length. In some embodiments, the blocked primers
comprise a 5' domain with a length between 25 and 60 nucleotides in
length and a 3' domain with a length between 14 and 30 nucleotides,
wherein the 5' domain is between 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 34, 35, 40, 45, 50 or 55 nucleotides on the low end of the
range, and 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55
or 60 nucleotides on the high end of the range and the 3' domain is
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or
30 nucleotides in length. In some embodiments, the blocked primers
comprise a 5' domain with a length between 25 and 60 nucleotides in
length and a 3' domain with a length between 15 and 30 nucleotides,
wherein the 5' domain is between 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 34, 35, 40, 45, 50 or 55 nucleotides on the low end of the
range, and 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55
or 60 nucleotides on the high end of the range and the 3' domain is
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
nucleotides in length.
In some embodiments, the blocked primers comprise a 5' domain with
a length between 15 and 30 nucleotides and a 3' domain with a
length between 14 and 30 nucleotides, wherein the 5' domain is 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30
nucleotides in length and the 3' domain is 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
In some embodiments, the blocked primers comprise a 5' domain that
is 30 nucleotides in length and a 3' domain that is 15 nucleotides
in length. See Example 2.
In some embodiments, the blocked primers comprise a 5' domain with
a length of 24 or 25 nucleotides, a ribobase such as rA or rG, a 3'
domain with a length of 5 or 6 nucleotides and a blocking moiety.
See FIG. 10 and V1 primer configuration. In some embodiments, the
blocked primers comprise a 5' domain with a length of 30, 31, 32,
33, 34 or 35 nucleotides, a ribobase such as rC or rU, a 3' domain
with a length of 15, 16 or 17 nucleotides and a blocking moiety.
See FIG. 10 and V2 primer configuration. In some embodiments, the
blocking primers comprise a 5' domain with a length of 31 or 35
nucleotides, a ribobase such as rC, a 3' domain with a length of 5
nucleotides and a blocking moiety. See FIG. 10 and V3 primer
configuration. In some embodiments, the blocked primers comprise a
5' domain with a length of 30 or 32 nucleotides, a ribobase such as
rU, a 3' domain with a length of 10 nucleotides and a blocking
moiety. See FIG. 10 and V4 primer configuration. In some
embodiments, the blocked primers comprise a 5' domain with a length
of 15 or 17 nucleotides, a ribobase such as rG or rU, a 3' domain
with a length of 15 nucleotides and a blocking moiety. See FIG. 10
and V5 primer configuration.
In some embodiments, the blocked primers comprise a 5' domain that
is at least 25 nucleotides and a 3' domain that is at least 14
nucleotides in length, wherein the 5' domain is between 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60,
65, 70, 80 or 90 nucleotides on the low end of the range, and 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55,
60, 65, 70, 80, 90, or 100 nucleotides on the high end of the range
and the 3' domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29 or 30 nucleotides in length. See primer
configuration V2 of Example 1. In some embodiments, the blocked
primers comprise a 5' domain that is 15 to 25 nucleotides and a 3'
domain that is at least 14 nucleotides in length, wherein the 5'
domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in
length and the 3' domain is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29 or 30 nucleotides in length. See primer
configuration V5 of Example 1.
In some embodiments, the blocked primers comprise a 5' domain that
is at least 25 nucleotides wherein, the 3' domain is 14
nucleotides; or the 3' domain is 15 nucleotides; or the 3' domain
is 16 nucleotides; or the 3' domain is 17 nucleotides; or the 3'
domain is 18 nucleotides; or the 3' domain is 19 nucleotides; or
the 3' domain is 20 nucleotides; or the 3' domain is 21
nucleotides; or the 3' domain is 22 nucleotides; or the 3' domain
is 23 nucleotides; or the 3' domain is 24 nucleotides; or the 3'
domain is 25 nucleotides; or the 3' domain is 26 nucleotides; or
the 3' domain is 27 nucleotides; or the 3' domain is 28
nucleotides; or the 3' domain is 29 nucleotides; or the 3' domain
is 30 nucleotides. In some embodiments, the blocked primers
comprise a 5' domain that is between 15 and 25 nucleotides in
length, wherein, the 3' domain is 14 nucleotides; or the 3' domain
is 15 nucleotides; or the 3' domain is 16 nucleotides; or the 3'
domain is 17 nucleotides; or the 3' domain is 18 nucleotides; or
the 3' domain is 19 nucleotides; or the 3' domain is 20
nucleotides; or the 3' domain is 21 nucleotides; or the 3' domain
is 22 nucleotides; or the 3' domain is 23 nucleotides; or the 3'
domain is 24 nucleotides; or the 3' domain is 25 nucleotides; or
the 3' domain is 26 nucleotides; or the 3' domain is 27
nucleotides; or the 3' domain is 28 nucleotides; or the 3' domain
is 29 nucleotides; or the 3' domain is 30 nucleotides.
In some embodiments, the blocked primers comprise a 5' domain that
is 10 to 100 nucleotides in length and a 3' domain that is 11 to 30
nucleotides in length. In some embodiments, the 5' domain is 15 to
50 nucleotides in length. In some embodiments, the 5' domain is 15
to 30 nucleotides. In some embodiments, the 3' domain is 14 to 25
nucleotides in length or 15 to 25 nucleotides length. In some
embodiments, the 3' domain is 14 to 20 nucleotides in length
wherein the ribobase is rU, rG or rA. In some embodiments, the 3'
domain is 15 to 20 nucleotides in length wherein the ribobase is
rU, rG or rA.
In some embodiments, the 3' domain optionally comprises a
mismatched base pair. In some embodiments, a 3' nucleotide of the
3' domain of a blocked forward primer is mismatched to a forward
primer binding sequence. In some embodiments, a 3' nucleotide of
the 3' domain of a blocked reverse primer is mismatched to a
reverse primer binding sequence. In some embodiments, the 3' domain
optionally comprises more than one mismatched base pair.
In some embodiments, the methods, compositions and kits described
herein for amplifying nucleic acid template, at least one blocked
primer is used wherein the 3' domain is from 10 to 30 nucleotides
in length. In some embodiments, the compositions and kits comprise
at least one blocked primer wherein the 3' domain is from 10 to 30
nucleotides in length.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits for amplifying nucleic
acids, a standard (non-blocked) primer can be used in combination
with at least one blocked primer. In some embodiments, the
non-blocked primers typically have a free 3' hydroxyl. It is
understood that use of the term "standard primer" refers to a
non-blocked primer (no ribobase cleavage location or blocking
moiety) and not a blocked primer of the invention. In some
embodiments, standard primers comprise polymers of
deoxyribonucleotides or analogs thereof. In some embodiments,
standard primers comprise naturally-occurring, synthetic,
recombinant, cloned, amplified, or unamplified forms. In some
embodiments, non-blocked primers include phosphodiester linkages
between all nucleotides.
In some embodiments, standard primers can be any length, including
about 5-100 nucleotides, or about 10-100 nucleotides, or about
15-100 nucleotides, or about 20-100 nucleotides, or longer.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits for amplifying nucleic
acids, wherein in addition to the blocked primers discussed above,
a reaction mixture is formed containing the necessary components
for amplification of the template nucleic acid. Those components,
in some embodiments, include one or more nucleic acid templates, a
polymerase, a recombinase, a recombinase accessory protein, a
forward primer, a reverse primer, dNTPs, an RNase H enzyme, a
buffer, and optionally a sieving agent, and optionally a crowding
agent, and optionally a single-stranded binding protein.
In some embodiments, methods for nucleic acid amplification can
include at least one co-factor for recombinase or polymerase
activity. In some embodiments, a co-factor comprises one or more
divalent cation. Examples of divalent cations include magnesium,
manganese and calcium. In some embodiments, the reaction mixture
comprises a buffer comprising a divalent cation. In embodiments the
buffer comprises magnesium or manganese ions.
In some embodiments, the reaction mixture may be formed by the
individual addition of each component to an aqueous or emulsion
solution. In some embodiments, the reaction mixture can be in the
form of a dehydrated pellet that requires rehydration prior to use.
In some embodiments, the reaction mixture is in the form of a
dehydrated pellet and comprises recombinase, recombinase accessory
proteins, gp32, DNA polymerase, dNTPs, ATP, phosphocreatine, a
crowding agent and creatine kinase. See Example 1. Rehydration
buffer can include, for example, Tris buffer, potassium acetate
salt and a crowding agent such as PEG.
In some embodiments, the reaction mixture is in the form of a
dehydrated pellet and comprises recombinase, recombinase accessory
protein(s), gp32, T7 DNA polymerase, thioredoxin, dNTPs, ATP,
phosphocreatine, a crowding agent and creatine kinase. In some
embodiments, when a dehydrated pellet is used that includes
reaction mixture components, the pellet is rehydrated with a
rehydration buffer, template DNA, primers including blocked primers
of the invention, RNase H enzyme and additional nuclease-free water
are added to a final volume.
In some embodiments, a nucleic acid amplification reaction can be
pre-incubated under conditions that inhibit premature reaction
initiation. For example, one or more components of a nucleic acid
amplification reaction can be withheld from a reaction vessel to
prevent premature reaction initiation. To start the reaction, a
divalent cation can be added (e.g., magnesium or manganese). In
another example, a nucleic acid amplification reaction can be
pre-incubated at a temperature that inhibits enzyme activity. The
reaction can be pre-incubated at about 0-15.degree. C., or about
15-25.degree. C. to inhibit premature reaction initiation. The
reaction can then be incubated at a higher temperature to induce
enzymatic activity. In some embodiments, the reaction mixture is
not exposed to a temperature above 40.degree. C. during the
amplification. Further details and examples of reaction mixtures
and components thereof, are found herein, for example in
discussions of composition embodiments as well as discussion herein
related to individual components of the reaction mixtures and
compositions.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits for amplifying nucleic
acids, wherein the nucleic acid templates (e.g., nucleic acid
templates) include a forward primer binding site having a forward
primer binding sequence and a reverse primer binding site having a
reverse primer binding sequence. In some embodiments, the primers
are referred to as a first and a second primer wherein the template
comprises a first primer binding sequence and a second primer
sequence. In embodiments the first and second primers are blocked
universal primers, such as 3' blocked universal primers that are
cleavable by RNase H. In some embodiments, the reaction mixture
comprises one monoclonal template nucleic acid. In some
embodiments, reaction mixture comprises at least two different
(polyclonal) polynucleotide or nucleic acid templates.
In some embodiments, the reaction mixture for the methods for
nucleic acid amplification comprise a plurality of different
polynucleotides. In some embodiments, a plurality of different
polynucleotides comprises single-stranded or double-stranded
polynucleotides, or a mixture of both. In some embodiments, a
plurality of different polynucleotides comprises polynucleotides
having the same or different sequences. In some embodiments, a
plurality of different polynucleotides comprises polynucleotides
having the same or different lengths. In some embodiments, a
plurality of different polynucleotides comprises about 2-10, or
about 10-50, or about 50-100, or about 100-500, or about 500-1,000,
or about 1,000-5,000, or about 10.sup.3-10.sup.6, or about
10.sup.6-10.sup.10 or more different polynucleotides. In some
embodiments, a plurality of different polynucleotides comprises
polymers of deoxyribonucleotides, ribonucleotides, and/or analogs
thereof. In some embodiments, a plurality of different
polynucleotides comprises naturally-occurring, synthetic,
recombinant, cloned, amplified, unamplified or archived (e.g.,
preserved) forms. In some embodiments, a plurality of different
polynucleotides comprises DNA, cDNA RNA or chimeric RNA/DNA, and
nucleic acid analogs.
In some embodiments, a plurality of different polynucleotide
templates amplified in methods provided herein can comprise a
double-stranded polynucleotide library construct having a nucleic
acid adaptor sequence on one or both ends. For example, a
polynucleotide library construct can comprise a first and second
end, where the first end is joined to a first nucleic acid adaptor.
A polynucleotide library construct can also include a second end
joined to a second nucleic acid adaptor. The first and second
adaptors can have the same or different sequence. In some
embodiments, at least a portion of the first or second nucleic acid
adaptor (i.e., as part of the polynucleotide library construct) can
hybridize to the first primer, which can be a universal primer. In
some embodiments, a homologous recombination enzyme, as part of a
nucleoprotein complex, can bind to a polynucleotide library
construct having a first or second nucleic acid adaptor
sequence.
In some embodiments, polynucleotide library constructs can be
compatible for use in any type of sequencing platform including
chemical degradation, chain-termination, sequence-by-synthesis,
pyrophosphate, massively parallel, ion-sensitive, single molecule
platforms, and combinations thereof.
In some embodiments, methods for nucleic acid amplification include
diluting the amount of polynucleotides that are reacted with beads
(e.g., beads attached with a plurality of a first primer, such as a
first RNase cleavable blocked primer of the invention) to reduce
the percentage of beads that react with more than one
polynucleotide. In some embodiments, nucleic acid amplification
reactions can be conducted with a polynucleotide-to-bead ratio that
is selected to optimize the percentage of beads having a monoclonal
population of polynucleotides attached thereto. For example, a
nucleic acid amplification reaction can be conducted at anyone of
polynucleotide-to-bead ratios in a range of about 1:1 or 1:2 to
1:500. In some embodiments, a polynucleotide-to-bead ratio includes
about 1:1, or about 1:2, or about 1:5, or about 1:10, or about
1:25, or about 1:50, or about 1:75, or about 1:100, or about 1:125,
or about 1:150, or about 1:175, or about 1:200, or about 1:225, or
about 1:225, or about 1:250. In some embodiments, a nucleic acid
amplification reaction can produce beads having zero types of
polynucleotides attached thereto, other beads having one type of
polynucleotide attached thereto, and other beads having more than
one type of polynucleotides attached thereto.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits for amplifying nucleic
acids, wherein the reaction mixtures comprise a recombinase.
Similarly, compositions and kits provided herein can include a
recombinase. The recombinase can include any agent that is capable
of inducing, or increasing the frequency of occurrence, of a
recombination event. A recombination event includes any event
whereby two different polynucleotides strands are recombined with
each other. Recombination can include homologous recombination. The
recombinase can be an enzyme, or a genetically engineered
derivative thereof. The recombinase optionally can associate with
(e.g., bind) a single-strand oligonucleotide (e.g., a first
primer). In some embodiments, an enzyme that catalyzes homologous
recombination can form a nucleoprotein complex by binding a
single-stranded oligonucleotide. In some embodiments, a homologous
recombination enzyme, as part of a nucleoprotein complex, can bind
a homologous portion of a double-stranded polynucleotide. In some
embodiments, the homologous portion of the polynucleotide can
hybridize to at least a portion of the first primer. In some
embodiment, the homologous portion of the polynucleotide can be
partially or completely complementary to at least a portion of the
first primer.
In some embodiments, a homologous recombination enzyme can catalyze
strand invasion by forming a nucleoprotein complex and binding to a
homologous portion of a double-stranded polynucleotide to form a
recombination intermediate having a triple-strand structure (D-loop
formation) (U.S. Pat. No. 5,223,414 to Zarling, U.S. Pat. Nos.
5,273,881 and 5,670,316 both to Sena, and U.S. Pat. Nos. 7,270,981,
7,399,590, 7,435,561, 7,666,598, 7,763,427, 8,017,339, 8,030,000,
8,062,850, and 8,071,308).
In some embodiments, the recombinase of the reaction mixtures,
compositions, and kits provided herein can include any suitable
agent that can promote recombination between polynucleotide
molecules. The recombinase can be an enzyme that catalyzes
homologous recombination. For example, the reaction mixture can
include a recombinase that includes, or is derived from, a
bacterial, eukaryotic or viral (e.g., phage) recombinase
enzyme.
In some embodiments, a homologous recombination enzyme comprises
wild-type, mutant, recombinant, fusion, or fragments thereof. In
some embodiments, a homologous recombination enzyme comprises an
enzyme from any organism, including myoviridae (e.g., uvsX from
bacteriophage T4, RB69, and the like) Escherichia coli (e.g.,
recA), or human (e.g., RAD51). In some embodiments, the reaction
mixture includes one or more recombinases selected from uvsX, RecA,
RadA, RadB, Rad51, a homologue thereof, a functional analog thereof
or a combination thereof. The recombinase in illustrative examples
is uvsX. The UvsX protein can be present, for example, at 50-250
ng/ul or 100-200 ng/ul.
In some embodiments, methods for nucleic acid amplification
comprise one or more accessory proteins. For example, an accessory
protein can improve the activity of a recombinase enzyme (U.S. Pat.
No. 8,071,308 granted to Piepenburg, et al.). In some embodiments,
an accessory protein can bind single strands of nucleic acids, or
can load a recombinase onto a nucleic acid. In some embodiments, an
accessory protein comprises wild-type, mutant, recombinant, fusion,
or fragments thereof. In some embodiments, accessory proteins can
originate from any combination of the same or different species as
the recombinase enzyme that are used to conduct a nucleic acid
amplification reaction. Accessory proteins can originate from any
bacteriophage including a myoviral phage. Examples of a myoviral
phage include T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage
133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4,
cyanophage S-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage
nt-1, phi-1, Rb16, Rb43, Phage 31, phage 44RR2.8t, Rb49, phage Rb3,
and phage LZ2. Accessory proteins can originate from any bacterial
species, including Escherichia coli, Sulfolobus (e.g., S.
solfataricus) or Methanococcus (e.g., M. jannaschii).
In some embodiments, methods for nucleic acid amplification can
include single-stranded binding proteins. Single-stranded binding
proteins include myoviral gp32 (e.g., T4 or RB69), Sso SSB from
Sulfolobus solfataricus, MjA SSB from Methanococcus jannaschii, or
E. coli SSB protein.
In some embodiments, methods for nucleic acid amplification can
include proteins that can improve recombinase loading onto a
nucleic acid. For example, a recombinase loading protein comprises
a UvsY protein (U.S. Pat. No. 8,071,308 granted to Piepenburg). In
some embodiments, the reaction mixture includes recombinase
accessory proteins. In some embodiments, the recombinase accessory
protein is uvsY. UvsY can be present, for example, at 20 ng/ul to
100 ng/ul.
In some embodiments, the reaction mixture used herein for nucleic
acid amplification may include at least one co-factor for
recombinase assembly on nucleic acids or for homologous nucleic
acid pairing. In some embodiments, a co-factor comprises any form
of ATP including ATP and ATP.gamma.S.
In some embodiments, methods for nucleic acid amplification can
include at least one co-factor that regenerates ATP. For example, a
co-factor comprises an enzyme system that converts ADP to ATP. In
some embodiments, a co-factor comprises phosphocreatine and
creatine kinase.
The reaction mixture further comprises nucleotides (dNTPs) for
strand extension of one or more nucleic acid templates, and in some
embodiments resulting in a clonal population of the template
nucleic acid sequence. In some embodiments, the nucleotides are not
extrinsically labeled. For example, the nucleotides can be
naturally occurring nucleotides, or synthetic analogs that do not
include fluorescent moieties, dyes, or other extrinsic optically
detectable labels. Optionally, the reaction mixture includes
nucleotides that are naturally occurring nucleotides. Optionally,
the nucleotides do not include groups that terminate nucleic acid
synthesis (e.g., dideoxy groups, reversible terminators, and the
like).
Optionally, the reaction mixture includes nucleotides that are
naturally occurring nucleotides. Optionally, the nucleotides do not
include groups that terminate nucleic acid synthesis (e.g., dideoxy
groups, reversible terminators, and the like). In some embodiments,
the nucleotides comprise a label or tag, described in more detail
below.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits for nucleic acid
amplification which include contacting (e.g., mixing) one or more
nucleic acid templates with one or more primers in the presence of
one or more enzymes capable of polymerization. In some embodiments,
the one or more enzymes capable of polymerization include at least
one polymerase and a recombinase. In some embodiments, the at least
one polymerase includes a thermostable or thermolabile polymerase.
In some embodiments, the at least one polymerase includes a
biologically active fragment of a DNA or RNA polymerase that
maintains sufficient catalytic activity to polymerize or
incorporate at least one nucleotide under any suitable conditions.
In one embodiment, the at least one polymerase comprises a mutated
DNA or RNA polymerase that maintains sufficient catalytic activity
to perform nucleotide polymerization under any suitable conditions.
In another embodiment, the at least one polymerase includes one or
more amino acid mutations that do not disrupt processivity of the
polymerase; and wherein the at least one polymerase having at least
one mutation maintains sufficient catalytic activity to perform
polymerization.
In some embodiments, a polymerase includes any enzyme, or fragment
or subunit of thereof, that can catalyze polymerization of
nucleotides and/or nucleotide analogs. In some embodiments, a
polymerase requires an extendible 3' end. For example, a polymerase
requires a terminal 3' OH of a nucleic acid primer to initiate
nucleotide polymerization. The polymerase can be other than a
thermostable polymerase. For example, the polymerase can be active
at 37.degree. C. and/or more active at 37.degree. C. than at
50.degree. C., 60.degree. C., 70.degree. C. or higher.
In some embodiments, a polymerase comprises any enzyme that can
catalyze the polymerization of nucleotides (including analogs
thereof) into a nucleic acid strand. Typically, but not necessarily
such nucleotide polymerization can occur in a template-dependent
fashion. In some embodiments, a polymerase can be a high fidelity
polymerase. Such polymerases can include without limitation
naturally occurring polymerases and any subunits and truncations
thereof, mutant polymerases, variant polymerases, recombinant,
fusion or otherwise engineered polymerases, chemically modified
polymerases, synthetic molecules or assemblies, and any analogs,
derivatives or fragments thereof that retain the ability to
catalyze such polymerization. Optionally, the polymerase can be a
mutant polymerase comprising one or more mutations involving the
replacement of one or more amino acids with other amino acids, the
insertion or deletion of one or more amino acids from the
polymerase, or the linkage of parts of two or more polymerases. The
term "polymerase" and its variants, as used herein, also refers to
fusion proteins comprising at least two portions linked to each
other, where the first portion comprises a peptide that can
catalyze the polymerization of nucleotides into a nucleic acid
strand and is linked to a second portion that comprises a second
polypeptide, such as, for example, a reporter enzyme or a
processivity-enhancing domain. Typically, the polymerase comprises
one or more active sites at which nucleotide binding and/or
catalysis of nucleotide polymerization can occur. In some
embodiments, a polymerase includes or lacks other enzymatic
activities, such as for example, 3' to 5' exonuclease activity or
5' to 3' exonuclease activity. In some embodiments, a polymerase
can be isolated from a cell, or generated using recombinant DNA
technology or chemical synthesis methods. In some embodiments, a
polymerase can be expressed in prokaryote, eukaryote, viral, or
phage organisms. In some embodiments, a polymerase can be
post-translationally modified proteins or fragments thereof.
In some embodiments, the polymerase can include any one or more
polymerases, or biologically active fragment of a polymerase, which
is described in U.S. Patent Publ. No. 2011/0262903 to Davidson et
al., published Oct. 27, 2011, and/or International PCT Publ. No. WO
2013/023176 to Vander Horn et al., published Feb. 14, 2013.
In some embodiments, a polymerase can be a DNA polymerase and
include without limitation bacterial DNA polymerases, eukaryotic
DNA polymerases, archaeal DNA polymerases, viral DNA polymerases
and phage DNA polymerases.
In some embodiments, a polymerase can be a replicase, DNA-dependent
polymerase, primases, RNA-dependent polymerase (including
RNA-dependent DNA polymerases such as, for example, reverse
transcriptases), a thermo-labile polymerase, or a thermo-stable
polymerase. In some embodiments, a polymerase can be any Family A
or B type polymerase. Many types of Family A (e.g., E. coli Pol I),
B (e.g., E. coli Pol II), C (e.g., E. coli Pol III), D (e.g.,
Euryarchaeotic Pol II), X (e.g., human Pol beta), and Y (e.g., E.
coli UmuC/DinB and eukaryotic RAD30/xeroderma pigmentosum variants)
polymerases are described in Rothwell and Watsman 2005 Advances in
Protein Chemistry 71:401-440. In some embodiments, a polymerase can
be a T3, T5, T7, or SP6 RNA polymerase.
In some embodiments, nucleic acid amplification reactions can be
conducted with one type or a mixture of polymerases, recombinases
and/or ligases. In some embodiments, nucleic acid amplification
reactions can be conducted with a low fidelity or high fidelity
polymerase.
In some embodiments, the reaction mixture can include a polymerase.
The polymerase optionally has, or lacks, exonuclease activity. In
some embodiments, the polymerase has 5' to 3' exonuclease activity,
3' to 5' exonuclease activity, or both. Optionally, the polymerase
lacks any one or more of such exonuclease activities.
In some embodiments, the polymerase has strand displacing activity.
Examples of useful strand-displacing polymerases include
Bacteriophage .PHI.29 DNA polymerase and Bst DNA polymerase.
An exemplary polymerase is Bst DNA Polymerase (Exonuclease Minus),
is a 67 kDa Bacillus stearothermophilus DNA Polymerase protein
(large fragment), exemplified in accession number 2BDP_A, which has
5'-3' polymerase activity and strand displacement activity but
lacks 3'-5' exonuclease activity. Other polymerases include Taq DNA
polymerase I from Thermus aquaticus (exemplified by accession
number 1TAQ), Eco DNA polymerase I from Escherichia coli (accession
number P00582), Aea DNA polymerase I from Aquifex aeolicus
(accession number 067779), or functional fragments or variants
thereof, e.g., with at least 80%, 85%, 90%, 95% or 99% sequence
identity at the nucleotide level.
In some embodiments, the DNA polymerase is a Bsu DNA polymerase
(large fragment (NEB). Bsu DNA Polymerase I, Large Fragment retains
the 5'.fwdarw.3' polymerase activity of the Bacillus subtilis DNA
polymerase I (1), but lacks the 5'.fwdarw.3' exonuclease domain. In
some embodiments, the Bsu DNA Polymerase large fragment lacks
3'.fwdarw.5' exonuclease activity. In some embodiments, Bsu DNA
Polymerase large fragment has optimal activity at 37.degree. C.
In one embodiment, the one or more enzymes capable of
polymerization include a T5 or T7 DNA polymerase. In some
embodiments, the one or more enzymes capable of polymerization
include a T5 or T7 DNA polymerase having one or more amino acid
mutations that reduce 3'-5' exonuclease activity. In some
embodiments, the T5 or T7 DNA polymerase having one or more amino
acid mutations that reduce 3'-5' exonuclease activity, does not
contain an amino acid mutation that disrupts processivity of the T5
or T7 DNA polymerase. In some embodiments, the T5 or T7 DNA
polymerase can include one or more amino acid mutations that
eliminate detectable 3'-5' exonuclease activity; and wherein the
one or more amino acid mutations do not disrupt processivity of the
T5 or T7 DNA polymerase. In some embodiments, the reaction mixture
comprises a Sau polymerase, T7 DNA polymerase with reduced 3' to 5'
exonuclease activity, Bsu polymerase, or a combination thereof.
In some embodiments, the one or more enzymes capable of
polymerization can include any suitable RNA polymerase. Suitable
RNA polymerases include, without limitation, T3, T5, T7, and SP6
RNA polymerases.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits, wherein the nucleic acid
amplification includes a combination of recombinase-polymerase
amplification (RPA) and the blocked primers under isothermal
conditions. During amplification, the blocked primers are cleaved
at the ribobase by a ribonuclease (RNase) to permit primer
extension and template amplification. As is known, RNases are
enzymes catalyzing hydrolysis of RNA into smaller components. The
use of RNase H enzyme with the blocked primers of the invention
provides advantages over methods of the art wherein their use
reduces: 1) nonspecific primer tailing via blocked 3' end; and 2)
non-templated primer dimer and nonspecific product formation, via
specific RNase H cleavage on RNA/RNA duplex between primer and
template strands.
In some embodiments, the compositions (e.g. reaction mixtures),
methods, and kits, include a ribo-endonuclease that is active at
appropriate temperatures for recombinase and polymerase activity
and are compatible with those enzymes. The ribo-endonuclease used
in the compositions, methods, and kits provided herein, can be an
RNase H enzyme, which represents a family of non-sequence specific
endonucleases that catalyze the cleavage of RNA via a hydrolytic
mechanism wherein the enzyme cleaves the 3'-O--P bond of RNA in a
DNA/RNA duplex substrate, provided that the RNase H enzyme is
active at appropriate temperatures for recombinase and polymerase
activity and is compatible with those enzymes.
The RNase H enzyme and its family of enzymes include two classes,
type 1 and type 2 RNase H based on the difference in their amino
acid sequence. Type 1 RNases H include prokaryotic and eukaryotic
RNases H1 and retroviral RNase H. Type 2 RNases H include
prokaryotic and eukaryotic RNases H2 and bacterial RNase H3. These
RNases H exist in a monomeric form, except for eukaryotic RNases
H2, which exist in a heterotrimeric form. All of these enzymes
share the characteristic that they are able to cleave the RNA
component of an RNA:DNA heteroduplex or within a DNA:DNA duplex
containing RNA base(s) within one or both of the strands. The
cleaved product yields a free 3'-OH for both classes of RNase H.
RNase H1 requires more than a single RNA base within an RNA:DNA
duplex for optimal activity, whereas RNase HII requires only a
single RNA base in an RNA:DNA duplex.
In some embodiments, the RNase H enzyme can be any RNase H enzymes,
provided that the enzyme retains sufficient activity at appropriate
temperatures for recombinase and polymerase activity and is
compatible with those enzymes. Therefore, the kits, compositions,
and methods can include an RNase H enzyme that has higher activity
at 37.degree. C. than it has at least one of the following
temperatures: 60.degree. C., 65.degree. C., 70.degree. C.,
75.degree. C., or 80.degree. C. For example, an RNase H enzyme used
in the kits, compositions, and methods herein can have a higher
activity at 37.degree. C. than at 75.degree. C. or have a higher
activity at 37.degree. C. than at 70.degree. C. In some
embodiments, the RNase H enzyme cleaves the oligonucleotide more
efficiently at 37.degree. C. than at 60.degree. C. wherein cleavage
of the ribobase present in the primer occurs at a temperature below
42.degree. C. Accordingly, in illustrative examples of any of the
embodiments provided herein, the RNase H enzyme is not a
thermostable RNase H enzyme (i.e. the RNase H enzyme is other than
a thermostable RNase H enzyme). In some embodiments, the RNase H
enzyme that has significant activity at 20 to 42.degree. C. The
methods, compositions, and kits provided herein can include in
illustrative examples, an RNase H enzyme that has significant
activity at 37.degree. C. In some embodiments, the RNase H has
sufficient activity to carry out the claim methods at 37.degree.
C.
As indicated, the RNase H enzyme included in the methods,
compositions, and kits provided herein can be any RNase H enzyme
that is active at appropriate temperatures for recombinase and
polymerase activity and is compatible with those enzymes. In some
embodiments, RNase H enzyme comprises RNase H1 (commercially
available from NEB, Inc.) or RNase H3. In alternative embodiments,
RNase H enzyme does not comprise RNase H1 or RNase H3. An exemplary
RNase H enzyme includes E. coli RNase HII (available for example
from NEB, Inc. (product M0288)). In some embodiments, the
endonuclease can be an RNase HII which cleaves a ribobase within a
DNA duplex and leaves a 3' hydroxyl end, and temperatures at which
it retains high activity are compatible with those of recombinase
and recombinase associated proteins. In some embodiments, the RNase
can be E. coli RNase H (available from NEB, Inc., for example
product M0297) (products/m0297-rnase-h), which also cleaves a
ribo-base when hybridized to DNA and leaves a 3'-hydroxyl end.
In some embodiments, the methods, as well as related compositions
and kits include a blocked primer design with 2-5 consecutive
ribobases. RNase H2 from Pyrococcus abyssi (P.a.), however, has low
activity at room temperature with optimal activity around
70.degree. C., a temperature above the range for the RPA methods.
Accordingly, in some embodiments herein, a higher temperature is
used for primer activation than used for amplification. For
example, a primer amplification step at between 42.degree. C. and
70.degree. C., 45.degree. C. and 70.degree. C., or 50.degree. C.
and 70.degree. C., or 50.degree. C. and 65.degree. C., or
60.degree. C. and 70.degree. C. can be performed, before an
amplification at temperatures disclosed for the amplification
methods herein, such as between 20.degree. C. and 42.degree. C.
Some embodiments include performing primer activation and
polymerization at two separate temperatures. RNase H2, such as
RNase HII, can be used in such 2-step methods as well, since it is
known to retain activity even at high temperatures.
The use of blocked primers are a potentially rate limiting step in
the RPA methods (See FIG. 1), because the 3' domain and block must
be removed before primer extension can proceed. To ensure the PRA
reaction proceeds rapidly, in some embodiments an excess (i.e.
non-limiting) amount of the RNase enzyme is used. One of skill
understands an excess can be determined empirically, see Example 3
for example. However in embodiments between 2.times., 3.times.,
4.times., 5.times., 6.times., 7.times., 8.times., 9.times.,
10.times., 11.times., 12.times., 13.times., 14.times., and
15.times. on the low end of the range, and 5.times., 6.times.,
7.times., 8.times., 9.times., 10.times., 11.times., 12.times.,
13.times., 14.times., 15.times., 16.times., 17.times., 18.times.,
19.times., 20.times. or 21.times., on the high end of the range
concentration of RNase H as compared to a minimally excess
concentration, are used in the RPA methods. The exact concentration
will depend on the starting concentration and other amplification
parameters. One unit of RNase is defined as the amount of enzyme
required to yield a fluorescence signal consistent with the nicking
of 100 picomol of synthetic double-stranded DNA (dsDNA) substrate
containing a single ribonucleotide near the quencher of a
fluorophore/quencher pair in 30 minutes at 37.degree. C. in
1.times. ThermoPol Buffer (NEB, Inc.). The dsDNA substrate can be a
26-mer present at 30 nM in a total reaction volume of 150 .mu.l as
indicated in the RNase HII product manual for RNase HII product
M0288 of NEB, Inc., and as used to determine unit activity or RNase
HII by NEB, Inc.
As described in Example 3, the RNase H enzyme concentration used in
the RPA amplification reaction can be characterized as a
"prohibiting", "limiting" or "excess" amount. Those concentrations
are determined empirically and may be different for different
blocked primer configurations, different concentration of starting
DNA template or different reaction times or temperature. In some
embodiments, an "excess" amount of RNase H enzyme when used with a
V2 or V5 primer configuration is 20 U or more in a 50 .mu.L
reaction volume. In some embodiments, a "limiting" amount of RNase
H enzyme is 5-10 U/50 .mu.L, such as 5, 6, 7, 8, 9, 10 U/50 .mu.L.
In some embodiments, a "limiting" amount of RNase H enzyme is less
than 20 U/50 .mu.L, such as 19, 18, 17, 16, 15, 14, 13, 12, 11, or
10 U/50 .mu.L. An "excess" amount of RNase H enzyme can be used in
any of the embodiments of the invention provided herein. Such
excess can be, for example, a concentration of equal to or greater
than 20 U/50 .mu.L (See FIGS. 8A and 8B).
In some embodiments, an E. coli RNase HII enzyme at a concentration
from 2.5 U to 200 U/50 .mu.L can be used. For example, an E. coli
RNase HII enzyme present in the reaction mixture at a concentration
from 5 to 150, 10 to 100, or 10 to 50 U/50 .mu.L can be used. In
some embodiments, greater than 10 U of RNase HII can be used.
In some embodiments, the RNase HII enzyme is present in the
reaction mixture at an excess concentration. For example, the RNase
HII enzyme can be an E. coli RNase HII enzyme and can be present at
a concentration from 20-250 U/50 .mu.L, 20-200 U/50 .mu.L, 20-150
U/50 .mu.L or 20-100 U/50 .mu.L. As a non-limiting, specific
example, the RNase H enzyme can be present in the reaction mixture
at a concentration of 20, 25, 30, 40, 50, 75, 100, 150, 200, or 250
U/50 .mu.L. The RNase H enzyme in certain examples of methods,
kits, compositions, and reaction mixtures provided herein, is
present at 2.times., 3.times., 4.times., 5.times., 10.times.,
20.times., 40.times. or 50.times. an excessive concentration. For
example, the RNase H enzyme can be an E. coli RNase HII enzyme
present at 200, 250, 300, 400, 500, 750, 1000, 1500, 2000, or 2500
U/50 .mu.L.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits, wherein the nucleic acid
amplification includes a reaction mixture which can include a
diffusion limiting agent. The diffusion limiting agent can be any
agent that is effective in preventing or slowing the diffusion of
one or more of the polynucleotide templates and/or one or more of
the amplification reaction products through the amplification
reaction mixture.
In some embodiments, the reaction mixture can include a sieving
agent. The sieving agent can be any agent that is effective in
sieving one or more polynucleotides present in the amplification
reaction mixture, such as for example amplification reaction
products and/or polynucleotide templates. In some embodiments, the
sieving agent restricts or slows the migration of polynucleotide
amplification products through the reaction mixture.
Inclusion of a sieving agent may be advantageous when clonally
amplifying two or more nucleic acid templates within a single
continuous liquid phase of a reaction mixture. For example, the
sieving agent can prevent or slow diffusion of templates, or
amplified polynucleotides produced via replication of at least some
portion of a template, within the reaction mixture, thus preventing
the formation of polyclonal contaminants without requiring
compartmentalization of the reaction mixture by physical means or
encapsulation means (e.g., emulsions) during the amplification.
Such methods of clonally amplifying templates within a single
continuous liquid phase of a single reaction mixture without need
for compartmentalization greatly reduces the cost, time and effort
associated with generation of libraries amenable for
high-throughput methods such as digital PCR, next generation
sequencing, and the like.
In some embodiments, the average pore size of the sieving agent is
such that movement of a target component within the reaction
mixture (e.g., a polynucleotide) is selectively retarded or
prevented. In one example, the sieving agent comprises any compound
that can provide a matrix having a plurality of pores that are
small enough to slow or retard the movement of a polynucleotide
through a reaction mixture containing the sieving agent. Thus, a
sieving agent can reduce Brownian motion of a polynucleotide.
In some embodiments, the amplification includes amplifying a
plurality of different polynucleotide templates onto a plurality of
different bead supports in the presence of a sieving agent, and
recovering a percentage of substantially monoclonal bead supports,
each such substantially monoclonal bead support include a bead
support attached to a substantially monoclonal polynucleotide
population. In some embodiments, the percentage of substantially
monoclonal bead supports recovered is substantially greater than
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%,
75%, 89%, 90%, or 95% of total amplified bead supports (i.e., total
bead supports including either polyclonal or monoclonal
populations) recovered from the reaction mixture. In some
embodiments, the percentage of substantially monoclonal bead
supports recovered is substantially greater than the percentage of
substantially monoclonal bead supports recovered following
amplification in the absence of the sieving agent but under
otherwise essentially similar or same amplification conditions.
In some embodiments, a sieving agent comprises a polymer compound.
In some embodiments, a sieving agent comprises a cross-linked or a
non-cross linked polymer compounds. By way of non-limiting
examples, the sieving agent can include polysaccharides,
polypeptides, organic polymers, etc.
In some embodiments, a sieving agent comprises linear or branched
polymers. In some embodiments, a sieving agent comprises charged or
neutral polymers.
In some embodiments, the sieving agent can include a blend of one
or more polymers, each having an average molecular weight and
viscosity.
In some embodiments, the sieving agent comprises a polymer having
an average molecular weight of about 10,000-2,000,000, or about
12,000-95,000, or about 13,000-95,000.
In some embodiments, a sieving agent can exhibit an average
viscosity range of about 5 centipoise to about 15,000 centipoise
when dissolved in water at 2 weight percent measured at about
25.degree. C., or about 10 centipoise to about 10,000 centipoise as
a 2% aqueous solutions measured at about 25.degree. C., or about 15
centipoise to about 5,000 centipoise as a 2% aqueous solution
measured at about 25.degree. C.
In some embodiments, a sieving agent comprises a viscosity average
molecular weight (M.sub.v) of about 25 to about 1,5000 kM.sub.v, or
about 75-1,000 kM.sub.v, or about 85-800 kM.sub.v. In some
embodiments, the reaction mixture comprises a sieving agent at
about 0.1 to about 20% weight per volume, or about 1-10% w/v, or
about 2-5% w/v.
In some embodiments, a sieving agent comprises a polysaccharide
polymer. In some embodiments, a sieving agent comprises a polymer
of glucose or galactose. In some embodiments, a sieving agent
comprises one or more polymers selected from the group consisting
of: cellulose, dextran, starch, glycogen, agar, chitin, pectin or
agarose. In some embodiments, the sieving agent comprises a
glucopyranose polymer.
In some embodiments, the sieving agent includes a cellulose
derivative, such as sodium carboxy methyl cellulose, sodium
carboxymethyl 2-hydroxyethyl cellulose, methyl cellulose, hydroxyl
ethyl cellulose, 2-hydroxypropyl cellulose, carboxy methyl
cellulose, hydroxyl propyl cellulose, hydroxyethyl methyl
cellulose, hydroxybutyl methyl cellulose, (hydroxypropyl)methyl
cellulose or hydroxyethyl ethyl cellulose, or a mixture including
any one or more of such polymers.
In some embodiments, the reaction mixture comprises a mixture of
different sieving agents, for example, a mixture of different
cellulose derivatives, starch, polyacrylamide, and the like.
In some embodiments, the reaction mixture can include a crowding
agent.
In some embodiments, the reaction mixture includes both a crowding
agent and a sieving agent.
In some embodiments, the reaction mixture includes at least one
diffusion-reducing agent. In some embodiments, a diffusion-reducing
agent comprises any compound that reduces migration of
polynucleotides from a region of higher concentration to one having
a lower concentration. In some embodiments, a diffusion reducing
agent comprises any compound that reduces migration of any
component of a nucleic acid amplification reaction irrespective of
size.
It should be noted that the concepts of a sieving agent and a
diffusion-reducing agent are not necessarily mutually exclusive; a
sieving agent can frequently be effective in reducing diffusion of
target compounds through a reaction mixture, whereas a diffusion
reducing agent can frequently have a sieving effect on reaction
components. In some embodiments, the same compound or reaction
mixture additive can act both as a sieving agent and/or a diffusion
reducing agent. Any of the sieving agents disclosed herein can in
some embodiments be capable of acting as a diffusion reducing agent
and vice versa.
In some embodiments, the diffusion reducing agent and/or sieving
agent includes polyacrylamide, agar, agarose or a cellulose polymer
such as hydroxyethyl cellulose (HEC), methyl-cellulose (MC) or
carboxymethyl cellulose (CMC).
In some embodiments, the sieving agent and/or the diffusion
reducing agent is included in the reaction mixture at
concentrations of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%,
40%, 50%, 74%, 90%, or 95% w/v (weight of agent per unit volume of
reaction mixture).
In some embodiments, the reaction mixture includes at least one
crowding agent. For example, a crowding agent can increase the
concentration of one or more components in a nucleic acid
amplification reaction by generating a crowded reaction
environment. In some embodiments, the reaction mixture includes
both a sieving agent and/or diffusion reagent and a crowding
agent.
In some embodiments, the nucleic acid amplification methods
comprise forming a reaction mixture by combining a nucleic acid
template having a forward primer binding sequence and a reverse
primer binding sequence, a polymerase, a recombinase, a
single-stranded binding protein, a recombinase loading protein, a
blocked forward primer, a blocked reverse primer, dNTPs, an RNase H
enzyme, and a buffer comprising a divalent cation. In some
embodiments, the forward primer binding sequence is complementary
or identical to at least a portion of the blocked forward primer
and the reverse primer binding sequence is complementary or
identical to at least a portion of the blocked reverse primer.
In some embodiments, the blocked forward primer and the blocked
reverse primer comprise a 5' domain and a 3' domain separated by a
nucleotide comprising a ribobase, wherein the 5' domain is 10 to 40
nucleotides in length and the 3' domain is 10 to 25 nucleotides in
length. In some embodiments, the blocked forward primer and the
blocked reverse primer comprise a 5' domain and a 3' domain
separated by a nucleotide comprising a ribobase, wherein the 5'
domain is 10 to 100 nucleotides in length and the 3' domain is 10
to 30 nucleotides in length.
In some embodiments, the reaction mixture further comprises a
recombinase accessory protein. In some embodiments, the recombinase
accessory protein is a single-stranded binding protein and/or a
recombinase loading protein.
In some embodiments, the reaction mixture comprises a blocked
primer wherein 5' domain is 15 to 30 nucleotides in length. In some
embodiments, the 5' domain of the blocked primers is 15 to 50
nucleotides in length. In some embodiments, the reaction mixture
includes a blocked primer wherein the 3' domain is 14 to 25
nucleotides in length. In some embodiments, the 3' domain is 15 to
25 nucleotides in length. In some embodiments, the 5' domain can be
at least 15 nucleotides and the 3' domain can be at least 10
nucleotides, wherein the length of the primer does not exceed 100,
90, 80, 75, 70, 60, or 50 nucleotides. In some embodiments, a 3'
nucleotide of the 3' domain of the forward primer is mismatched to
the forward primer binding sequence.
In some embodiments, the ribobase separating the 5' domain and the
3' domain of the blocked primer comprises rU, rG or rA. In some
embodiments, the ribobase separating the 5' domain and the 3'
domain of the blocked primer comprises rC. In some embodiments, the
3' domain of the blocked primers is 14 to 20 nucleotides in length
and the ribobase is rU, rG, rC or rA.
In some embodiments, the reaction mixture comprises a recombinase
accessory protein that is uvsY. In some embodiments, the reaction
mixture comprises a recombinase selected from the group consisting
of uvsX, RecA, RadA, RadB, Rad 51, a homologue thereof, a
functional analog thereof and a combination thereof. In some
embodiments, the reaction mixture comprises uvsY recombinase
accessory protein and uvsX recombinase.
In some embodiments, the reaction mixture comprises RNase H enzyme
that is RNase HII. In some embodiments, the RNase H enzyme is
present at a concentration from 20 U to 100 U/50 .mu.L. In some
embodiments, the RNase H enzyme is present at a concentration from
40 to 90 U/50 .mu.L.
In some embodiments, the nucleic acid template is a member of a
nucleic acid library comprising a population of nucleic acid
templates each comprising a forward primer binding sequence, and
wherein the blocked forward primer is a blocked universal forward
primer that binds the universal forward primer binding sequence. In
some embodiments, the nucleic acid templates each comprises a
reverse universal primer binding sequence and wherein the blocked
reverse primer is a blocked universal reverse primer that binds the
universal reverse primer binding sequence.
In some embodiments, either or both of the blocked forward primer
and the blocked reverse primer are immobilized on a solid support.
In some embodiments, the solid support is a bead.
In some embodiments, the nucleic acid amplification methods
comprise forming a reaction mixture by combining at least two
different polynucleotide templates comprising both a first primer
binding sequence and a second primer binding sequence, a
recombinase, a recombinase accessory protein, a polymerase, a first
blocked universal primer, a second blocked universal primer, dNTPs,
an RNase H enzyme, and a buffer. The reaction mixture is in contact
with a support having the first blocked universal primer bound
thereto, wherein the first primer binding sequence is complementary
or identical to at least a portion of the first blocked universal
primer and the second primer binding sequence is complementary or
identical to at least a portion of the second blocked universal
primer.
In some embodiments, at least two substantially monoclonal nucleic
acid populations are formed by using the polymerase to amplify each
of said at least two different polynucleotide templates onto
different sites on the solid support under substantially isothermal
conditions.
In some embodiments, the second blocked universal primer is in
solution (e.g., soluble primers). In some embodiments, the second
blocked universal primer is immobilized on the support.
In some embodiments, the reaction mixture and the at least two
substantially monoclonal nucleic acid populations are formed in the
same single continuous liquid phase. In some embodiments, the
reaction mixture and the at least two substantially monoclonal
nucleic acid populations are formed in a water-in-oil emulsion.
In some embodiments, the at least two different polynucleotide
templates are members of a polynucleotide library, wherein each
member of the polynucleotide library comprises the first primer
binding sequence and the second primer binding sequence. In some
embodiments, nucleic acids of the at least two substantially
monoclonal nucleic acid populations are sequenced.
In some embodiments, the first blocked universal primer and the
second blocked universal primer comprise a 5' domain and a 3'
domain separated by a nucleotide comprising a ribobase, wherein the
5' domain is 10 to 40 nucleotides in length and the 3' domain is 10
to 25 nucleotides in length. In some embodiments, the reaction
mixture comprises a first and second blocked primer wherein the 5'
domain is 15 to 30 nucleotides in length. In some embodiments, the
5' domain of the blocked primers is 15 to 50 nucleotides in length.
In some embodiments, the reaction mixture comprises a blocked
primer wherein the 3' domain is 14 to 25 nucleotides in length. In
some embodiments, the 3' domain is 15 to 25 nucleotides in length.
In some embodiments, a 3' nucleotide of the 3' domain of the first
primer is mismatched to the first primer binding sequence.
In some embodiments, the ribobase separating the 5' domain and the
3' domain of the blocked primer comprises rU, rG or rA. In
embodiments the ribobase separating the 5' domain and the 3' domain
of the blocked primer comprises rC. In some embodiments, the 3'
domain of the blocked primers is 14 to 20 nucleotides in length and
the ribobase is rU, rG, rC or rA.
In some embodiments, the reaction mixture comprises a recombinase
accessory protein that is uvsY. In some embodiments, the reaction
mixture comprises a recombinase selected from the group consisting
of uvsX, RecA, RadA, RadB, Rad 51, a homologue thereof, a
functional analog thereof and a combination thereof. In some
embodiments, the reaction mixture comprises uvsY recombinase
accessory protein and uvsX recombinase.
In some embodiments, the reaction mixture comprises RNase H enzyme
that is RNase HII. In some embodiments, the RNase H enzyme is
present at a concentration from 20 U to 100 U/50 .mu.L. In some
embodiments, the RNase H enzyme is present at a concentration from
40 to 90 U/50 .mu.L.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits, wherein the nucleic acid
amplification includes the amplification reaction mixture which can
include one or more solid or semi-solid supports. In some
embodiments, at least one of the supports can include one or more
instances of a first blocked primer including a first primer
sequence. As used herein, the blocked primers refer to those
described above containing a 5' domain, at least one ribobase, a 3'
domain and a blocking group. The appropriate length for the 5' and
3' domain the primers are described in detail above, for example
the 3' domain has a length of at least 10 nucleotides such as 10 to
30 nucleotides. In the section that follows, the primers that may
be attached to a support or surface are those blocked primers
described herein. For example, a forward or reverse blocked primer
is attached to a solid support via the 5' end of the primer. See
FIG. 11 for exemplary blocked primers for use on a solid
support.
In some embodiments, at least one polynucleotide template in the
reaction mixture includes a first primer binding sequence. The
first primer binding sequence can be substantially identical or
substantially complementary to the first blocked primer sequence.
In some embodiments, at least one, some or all of the supports
include a plurality of first blocked primers that are substantially
identical to each other. In some embodiments, all of the blocked
primers on the supports are substantially identical to each other,
or all include a substantially identical first primer sequence.
In some embodiments, at least one of the supports includes two or
more different blocked primers attached thereto. For example, the
at least one support can include at least one instance of the first
blocked primer and at least one instance of a second blocked
primer.
In some embodiments, the aqueous phase of the reaction mixture
includes a plurality of supports, at least two supports of the
plurality being attached to blocked primers including a first
priming sequence. In some embodiments, the reaction mixture
includes two or more different polynucleotide templates having a
first primer binding sequence.
Alternatively, in some embodiments, the reaction mixture does not
include any supports. In some embodiments, the at least two
different polynucleotide templates are amplified directly onto a
surface of the site or reaction chamber of the array. In some
embodiments, the reaction chambers are arranged in an array on a
support and the reaction chambers are used to conduct sequencing
reactions. In some embodiments, the reaction chambers are arranged
in an array on a sequencing support.
In some embodiments, methods for nucleic acid amplification
comprise one or more surfaces. In some embodiments, a surface can
be attached with a plurality of first primers, the first primers of
the plurality sharing a common first primer sequence.
In some embodiments, a surface can be an outer or top-most layer or
boundary of an object. In some embodiments, a surface can be
interior to the boundary of an object.
In some embodiments, the reaction mixture includes multiple
different surfaces, for example, the reaction mixture can include a
plurality of beads (such as particles, nanoparticles,
microparticles, and the like) and at least two different
polynucleotide templates can be clonally amplified onto different
surfaces, thereby forming at least two different surfaces, each of
which is attached to an amplicon. In some embodiments, the reaction
mixture includes a signal surface (for example, the surface of a
slide or array of reaction chambers) and at least two different
polynucleotide templates are amplified onto two different regions
or locations on the surface, thereby forming a single surface
attached to two or more amplicons.
In some embodiments, a surface can be porous, semi-porous or
non-porous. In some embodiments, a surface can be a planar surface,
as well as concave, convex, or any combination thereof. In some
embodiments, a surface can be a bead, particle, microparticle,
sphere, filter, flowcell, well, groove, channel reservoir, gel or
inner wall of a capillary. In some embodiments, a surface includes
the inner walls of a capillary, a channel, a well, groove, channel,
reservoir. In some embodiments, a surface can include texture
(e.g., etched, cavitated, pores, three-dimensional scaffolds or
bumps).
In some embodiments, a surface can be magnetic or paramagnetic bead
(e.g., magnetic or paramagnetic nanoparticles or microparticles).
In some embodiments, paramagnetic microparticles can be
paramagnetic beads attached with streptavidin (e.g., Dynabeads.TM.
M-270 from Invitrogen, Carlsbad, Calif.). Particles can have an
iron core, or comprise a hydrogel or agarose (e.g.,
Sepharose.TM.).
In some embodiments, the surface can have immobilized thereon, a
plurality of an RNase-cleavable first blocked primer. See Example
5. A surface can be coated with an acrylamide, carboxylic or amine
compound for attaching a nucleic acid (e.g., a first primer). In
some embodiments, an amino-modified nucleic acid (e.g., primer) can
be attached to a surface that is coated with a carboxylic acid. In
some embodiments, an amino-modified nucleic acid can be reacted
with EDC (or EDAC) for attachment to a carboxylic acid coated
surface (with or without NHS). A first blocked primer can be
immobilized to an acrylamide compound coating on a surface.
Particles can be coated with an avidin-like compound (e.g.,
streptavidin) for binding biotinylated nucleic acids.
In some embodiments, the surface comprises the surface of a bead.
In some embodiments, a bead comprises a polymer material. For
example, a bead comprises a gel, hydrogel or acrylamide polymers. A
bead can be porous. Particles can have cavitation or pores, or can
include three-dimensional scaffolds. In some embodiments, particles
can be Ion Sphere.TM. particles.
In some embodiments, the disclosed methods (as well as related
compositions, systems and kits) include immobilizing one or more
nucleic acid templates onto one or more supports. Nucleic acids may
be immobilized on the solid support by any method including but not
limited to physical adsorption, by ionic or covalent bond
formation, or combinations thereof. A solid support may include a
polymeric, a glass, or a metallic material. Examples of solid
supports include a membrane, a planar surface, a microtiter plate,
a bead, a filter, a test strip, a slide, a cover slip, and a test
tube. A solid support means any solid phase material upon which an
oligomer is synthesized, attached, ligated or otherwise
immobilized. A support can optionally comprise a "resin", "phase",
"surface" and "support". A support may be composed of organic
polymers such as polystyrene, polyethylene, polypropylene,
polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as
co-polymers and grafts thereof. A support may also be inorganic,
such as glass, silica, controlled-pore-glass (CPG), or
reverse-phase silica. The configuration of a support may be in the
form of beads, spheres, particles, granules, a gel, or a surface.
Surfaces may be planar, substantially planar, or non-planar.
Supports may be porous or non-porous, and may have swelling or
non-swelling characteristics. A support can be shaped to comprise
one or more wells, depressions or other containers, vessels,
features or locations. A plurality of supports may be configured in
an array at various locations. A support is optionally addressable
(e.g., for robotic delivery of reagents), or by detection means
including scanning by laser illumination and confocal or deflective
light gathering. An amplification support (e.g., a bead) can be
placed within or on another support (e.g., within a well of a
second support).
In some embodiments, the solid support is a "microparticle,"
"bead," "microbead," etc., (optionally but not necessarily
spherical in shape) having a smallest cross-sectional length (e.g.,
diameter) of 50 microns or less, preferably 10 microns or less, 3
microns or less, approximately 1 micron or less, approximately 0.5
microns or less, e.g., approximately 0.1, 0.2, 0.3, or 0.4 microns,
or smaller (e.g., under 1 nanometer, about 1-10 nanometer, about
10-100 nanometers, or about 100-500 nanometers). Microparticles
(e.g., Dynabeads from Dynal, Oslo, Norway) may be made of a variety
of inorganic or organic materials including, but not limited to,
glass (e.g., controlled pore glass), silica, zirconia, cross-linked
polystyrene, polyacrylate, polymethylmethacrylate, titanium
dioxide, latex, polystyrene, etc. Magnetization can facilitate
collection and concentration of the microparticle-attached reagents
(e.g., polynucleotides or ligases) after amplification, and can
also facilitate additional steps (e.g., washes, reagent removal,
etc.). In some embodiments of the invention a population of
microparticles having different shapes sizes and/or colors can be
used. The microparticles can optionally be encoded, e.g., with
quantum dots such that each microparticle can be individually or
uniquely identified.
In some embodiments, a bead surface can be functionalized for
attaching a plurality of a first blocked primer. In some
embodiments, a bead can be any size that can fit into a reaction
chamber. For example, one bead can fit in a reaction chamber. In
some embodiments more than one bead can fit in a reaction chamber.
In some embodiments, the smallest cross-sectional length of a bead
(e.g., diameter) can be about 50 microns or less, or about 10
microns or less, or about 3 microns or less, approximately 1 micron
or less, approximately 0.5 microns or less, e.g., approximately
0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer,
about 1-10 nanometer, about 10-100 nanometers, or about 100-500
nanometers). In some embodiments, a bead can be attached with a
plurality of one or more different blocked primer sequences. In
some embodiments, a bead can be attached with a plurality of one
blocked primer sequence, or can be attached a plurality of two or
more different blocked primer sequences. In some embodiments, a
bead can be attached with a plurality of at least 1,000 primers, or
about 1,000-10,000 primers, or about, 10,000-50,000 primers, or
about 50,000-75,000 primers, or about 75,000-100,000 primers, or
more. In some embodiments, the disclosure relates generally to
methods, as well as related compositions and kits, wherein the
nucleic acid amplification includes the reaction mixtures discussed
in the context of methods provided herein themselves form
embodiments of the invention. In some embodiments, the compositions
that include a recombinase, a polymerase suitable for RPA, and an
RNase H enzyme that is active at temperatures at which the
recombinase and polymerase are active, and is compatible with those
enzymes. In some embodiments, the compositions can further include
a single stranded binding protein and/or a recombinase loading
protein. The recombinase and polymerase are typically present at
effective concentrations for recombinase polymerase amplification,
or at higher concentrations such that they can be combined with
other reaction components into a final blocked primer-RPA reaction
mixture. RNase is present at an effective concentration, such as a
limiting and/especially an excess concentration, as disclosed
herein, or 2.times., 3.times., 4.times., 5.times., or 10.times.
such concentrations. The RNase can be any of the RNases discussed
herein, including in illustrative examples, E. coli RNase HII.
In some embodiments, the compositions can further include other
components of an RPA and/or RNase reaction. For example, the
compositions can include dNTPs and a buffer. In addition, the
composition can include a blocked forward primer and a blocked
reverse primer. As a non-limiting example, the composition can
include a recombinase, a polymerase, an RNase H enzyme that is
other than a thermostable RNase H, a nucleic acid template, uvsX
recombinase, uvsY recombinase loading protein, gp32 protein, Sau
DNA polymerase, dNTPs, ATP, phosphocreatine and creatine kinase. In
some embodiments, the composition can be in liquid form, or it can
be in a solid form, such as a dried-down pellet form that can be
rehydrated. Furthermore, components for compositions provided
herein, can be split up such that any combination of the components
can be in a pellet or liquid form, and one or more combinations of
the rest of the components can be in one or more separate pellet or
liquid forms. Such combinations can form kits that include at least
two of such combinations. For example, a kit of the invention can
include a pellet that includes all the reaction mixture components
provided herein except for the RNase enzyme, which can be provided
in a separate pellet or liquid in the kit.
In some embodiments, provided herein are compositions and kits
including at least one blocked primer of the invention that
includes a ribonucleotide as disclosed in detail herein. The
compositions and kits can further include a second blocked primer
or a standard primer. In some embodiments, the compositions and
kits include a pair of primers (forward and reverse) wherein at
least one is a blocked primer of the invention.
In some embodiments, the composition includes a reaction mixture
having at least a blocked primer that includes a ribonucleotide as
discussed in detail herein, and a recombinase. In some embodiments,
the composition further includes amplification reagents including
template nucleic acid, polymerase, RNase H, and/or accessory
proteins. The reaction mixture for an amplification reaction
typically includes a source of nucleotides, or analogs thereof,
that is used by the polymerase as substrates for an extension
reaction.
In some embodiments, a composition comprises nucleic acid template,
a polymerase, a recombinase, a blocked forward primer, a blocked
reverse primer, dNTPs, an RNase H enzyme, and a buffer. In some
embodiments, the composition comprises a nucleic acid template,
blocked forward primer, a blocked reverse primer, uvsX recombinase,
uvsY recombinase loading protein, gp32 protein, Sau DNA polymerase,
dNTPs, ATP, phosphocreatine and creatine kinase.
In some embodiments, the blocked forward primer and/or the blocked
reverse primer in compositions, reaction mixtures, and kits of the
invention can include any of the blocked primers disclosed herein.
For example, the blocked forward primer and/or the blocked reverse
primer can include a 5' domain and a 3' domain separated by a
nucleotide comprising a ribobase, wherein the 5' domain is 10 to 40
nucleotides in length and the 3' domain is 11 to 25 nucleotides in
length. In some embodiments, the composition comprises a forward
and reverse blocked primer wherein the 5' domain is 15 to 30
nucleotides in length. In some embodiments, the 5' domain of the
blocked primers is 15 to 50 nucleotides in length. In some
embodiments, the composition comprises a blocked primer wherein the
3' domain is 14 to 25 nucleotides in length. In some embodiments,
the 3' domain is 15 to 25 nucleotides in length. In some
embodiments, a 3' nucleotide of the 3' domain of the forward primer
is mismatched to the forward primer binding sequence.
In some embodiments, a composition of the invention comprises at
least two different polynucleotide templates comprising both a
first primer binding sequence and a second primer binding sequence,
a recombinase, a recombinase accessory protein, a polymerase, a
first blocked universal primer, a second blocked universal primer,
dNTPs, an RNase H enzyme, and a buffer. In some embodiments, the
composition further comprises a support. In some embodiments, the
support is a bead. In further embodiments, the first blocked
universal primer is attached to the bead support.
In some embodiments, the composition includes at least two
different polynucleotide templates comprising both a first primer
binding sequence and a second primer binding sequence, uvsX
recombinase, uvsY recombinase loading protein, gp32 protein, Sau
DNA polymerase, APT, phosphocreatine, creatine kinase, a first
blocked universal primer attached to a bead support, a second
blocked universal primer, an RNase H enzyme, and a buffer.
In some embodiments, the first blocked universal primer and the
second blocked universal primer comprise a 5' domain and a 3'
domain separated by a nucleotide comprising a ribobase, wherein the
5' domain is 10 to 40 nucleotides in length and the 3' domain is 10
to 25 nucleotides in length. In some embodiments, the composition
comprises a first and second blocked primer wherein the 5' domain
is 15 to 30 nucleotides in length. In some embodiments, the 5'
domain of the blocked primers is 15 to 50 nucleotides in length. In
some embodiments, the composition comprises a blocked primer
wherein the 3' domain is 14 to 25 nucleotides in length. In some
embodiments, the 3' domain is 15 to 25 nucleotides in length. In
some embodiments, a 3' nucleotide of the 3' domain of the first
primer is mismatched to the first primer binding sequence.
In some embodiments, compositions are also amendable to kit format
wherein the primers, and amplification may be in the same contain,
separate contains and in liquid or dehydrated form. The kit may
comprise instructions for performing the RPA methods for
amplification of nucleic acid template including clonal
amplification for downstream sequencing methods. In one embodiment,
the kit provides instructions for nucleic acid sequencing
preparation.
In some embodiments, provided herein is a kit that includes at
least two containers at least one of which includes a blocked, at
least one of which includes a recombinase and at least one of which
includes an RNase H. The recombinase, RNase H and the blocked can
be in the same or different tubes.
In some embodiments, at least one blocked primer can be attached to
a support. In some embodiments, the kit comprises at least one
blocked primer attached to a bead support.
In some embodiments, the container comprising the recombinase
further comprises one or more amplification reagents including a
recombinase accessory protein, a polymerase, dNTPs, an RNase H
enzyme, and a buffer. In some embodiments the kit comprises one or
more containers comprising uvsX recombinase, uvsY recombinase
loading protein, gp32 protein, Sau DNA polymerase, dNTPs, RNase H,
ATP, phosphocreatine and creatine kinase.
In some embodiments, the kit comprises a blocked forward primer and
a blocked reverse primer comprising a 5' domain and a 3' domain
separated by a nucleotide comprising a ribobase, wherein the 5'
domain is 10 to 40 nucleotides in length and the 3' domain is 10 to
25 nucleotides in length. In some embodiments, the kit comprises a
forward and reverse blocked primer wherein the 5' domain is 15 to
30 nucleotides in length. In some embodiments, the 5' domain of the
blocked primers is 15 to 50 nucleotides in length. In some
embodiments, the kit comprises a blocked primer wherein the 3'
domain is 14 to 25 nucleotides in length. In some embodiments, the
3' domain is 15 to 25 nucleotides in length. In some embodiments, a
3' nucleotide of the 3' domain of the forward primer is mismatched
to the forward primer binding sequence.
In some embodiments, the kit the first blocked universal primer and
the second blocked universal primer comprise a 5' domain and a 3'
domain separated by a nucleotide comprising a ribobase, wherein the
5' domain is 10 to 40 nucleotides in length and the 3' domain is 10
to 25 nucleotides in length. In some embodiments, the composition
comprises a first and second blocked primer wherein the 5' domain
is 15 to 30 nucleotides in length. In some embodiments, the 5'
domain of the blocked primers is 15 to 50 nucleotides in length. In
some embodiments, the kit comprises a blocked primer wherein the 3'
domain is 14 to 25 nucleotides in length. In some embodiments, the
3' domain is 15 to 25 nucleotides in length. In some embodiments, a
3' nucleotide of the 3' domain of the first primer is mismatched to
the first primer binding sequence.
In some embodiments, the kit comprises a first blocked universal
primer attached to a bead support. In some embodiments, the kit
further comprises instructions for clonal amplification of two or
nucleic acid templates to be used for nucleic acid sequencing.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions, kits, systems and apparatuses, for
nucleic acid amplification, comprising amplifying a nucleic acid
template to produce an amplicon using the RNase-cleavable blocked
primers disclosed herein. In some embodiments, the amplicon is a
substantially monoclonal population of polynucleotides.
Monoclonality can be desirable in nucleic acid assays because the
different characteristics of the diverse polynucleotides within a
polyclonal population can complicate the interpretation of assay
data. One example involves nucleic acid sequencing applications, in
which the presence of polyclonal populations can complicate the
interpretation of sequencing data; however, with many sequencing
systems are not sensitive enough to detect nucleotide sequence data
from a single polynucleotide template, thus requiring clonal
amplification of templates prior to sequencing.
In some embodiments, the amplification methods can be employed to
clonally amplify two or more different nucleic acid templates,
optionally using and within the same reaction mixture, to produce
at least two substantially monoclonal, and in some embodiments,
monoclonal nucleic acid populations. Optionally, at least one of
the substantially monoclonal populations is formed via
amplification of a single polynucleotide template.
In some embodiments, the reaction mixture can be incubated under
substantially isothermal amplification conditions thereby
amplifying the nucleic acid template(s). In some embodiments, the
isothermal conditions are typically between 20.degree. C. to
50.degree. C., in some embodiments 20.degree. C. to 45.degree. C.,
in some embodiments, 20.degree. C. to 45.degree. C., in other
embodiments, 25.degree. C. to 40.degree. C., and still other
embodiments 25.degree. C. to 37.degree. C. for 2 to 240 minutes. In
some embodiments, the temperature is between 30.degree. C. and
42.degree. C. In some embodiments, the reaction mixture is not
exposed to a temperature above 40.degree. C., or above 41.degree.
C., or above 42.degree. C., or above 43.degree. C., or above
45.degree. C. or not exposed to a temperature above 50.degree. C.
In some embodiments, the reaction mixture is not exposed to hot
start conditions. A rate limiting enzyme may be RNase H, wherein a
high concentration or excess (i.e. non-limiting) amount of the
endonuclease ensures the amplification reaction proceeds based on
the kinetics of the polymerase. See Example 3.
As illustrated in FIG. 1, once blocked primers hybridize to
complementary template sequences, RNase H enzyme is activated,
cleaving the ribonucleotide linkage in the blocked primer present
in duplex DNA. The 3' domain comprising the blocking group
dissociates, liberating the blocking group which blocks
amplification, creating a free 3'-hydroxyl which is now capable of
primer extension. Alternatively, RNase nicks the DNA, and the 3'
domain comprising the blocking group is displaced by the 5' domain
primer extension. RNase H enzyme used here is active at between
about 20.degree. C. to 45.degree. C., the temperature range for the
RPA amplification methods. One drawback to the use of recombinase
amplification methods is that primer/primer hybrids are typically
stable at these temperatures, leading to primer artifact
amplification. However, the use of the blocked primers comprising a
ribonuclease cleavage location reduces or eliminates primer dimer
product amplification. See Example 2.
In some embodiments, the amplification is typically performed under
substantially isothermal amplification conditions. The
substantially isothermal temperature can be between 20, 21, 22, 23,
24, 25, 30, 35 or 40 on the low end of the range, and 21, 22, 23,
24, 25, 26, 30, 35, 40 or 45 on the high end of the range. In some
embodiments, the temperature is between 20.degree. C. and
45.degree. C. In some embodiments, the temperature is between
35.degree. C. and 45.degree. C. In some embodiments, the
temperature is 37.degree. C.
In some embodiments, an isothermal RPA nucleic acid amplification
reaction can be conducted at about 15-25.degree. C., or about
25-35.degree. C., or about 35-40.degree. C., or about 35-42.degree.
C., or about 40-45.degree. C., or about 45-50.degree. C., or about
50-55.degree. C., or about 55-60.degree. C. However, it is
understood the enzymes used at these temperatures will need to be
optimized in combination and may require changes in the enzyme, for
example the DNA polymerase used, Bst instead of Bsu, or the RNase H
enzyme, such as RNase H2 instead of RNase HII.
In some embodiments, any of the nucleic acid amplification methods
disclosed herein can be conducted, or can include steps that are
conducted, under isothermal or substantially isothermal
amplification conditions. In some embodiments isothermal
amplification conditions comprise a nucleic acid amplification
reaction subjected to a temperature variation which is constrained
within a limited range during at least some portion of the
amplification (or the entire amplification process), including for
example a temperature variation is equal or less than about
20.degree. C., or about 10.degree. C., or about 5.degree. C., or
about 1-5.degree. C., or about 0.1-1.degree. C., or less than about
0.1.degree. C., or, for example a temperature variation is equal or
less than 20.degree. C., or 10.degree. C., or 5.degree. C., or
1-5.degree. C., or 0.1-1.degree. C., or less than 0.1.degree.
C.
The amplification can be carried out for 2 minutes to 240 minutes,
thereby amplifying the nucleic acid template. In some embodiments,
the reaction time is between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 50, 60, 70, 80, 90,
100, 120, 140, 160, 180, 200 or 220 minutes on the low end of the
range, and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 26, 27, 28, 29, 30, 25, 40, 50, 60, 70, 80, 90, 100,
120, 140, 160, 180, 200, 220 or 240 minutes on the high end of the
range. In some embodiments, the reaction mixture is incubated to
generate an amplified template for at least 5 minutes, wherein
reaction time is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 25, 40, 50, 60, 70, 80, 90, 100, 120, 140,
160, 180, 200 or 220 minutes on the low end of the range, and 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28,
29, 30, 25, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,
220 or 240 minutes on the high end of the range.
In some embodiments, the reaction mixture is incubated from 5 to 60
minutes. In some embodiments, the amplifying time is from 15 to 60
minutes. In some embodiments, the amplifying time is from 15 to 45
minutes. In some embodiments, the reaction mixture is incubated for
30 minutes to generate an amplified template sequence. In some
embodiments, the reaction mixture is incubated for 50 minutes to
generate an amplified template sequence.
In some embodiments, an isothermal nucleic acid amplification
reaction can be conducted for about 2, 5, 10, 15, 20, 30, 40, 50,
60 or 120 minutes.
In some embodiments, the reaction mixture is formed by combining a
nucleic acid template having a forward primer binding sequence and
a reverse primer binding sequence, with the following optional
reagents: a polymerase, a recombinase, a single-stranded binding
protein, a recombinase loading protein, a blocked forward primer, a
blocked reverse primer, dNTPs, ATP, phosphocreatine and creatine
kinase, an RNase H enzyme, and a buffer, and wherein a divalent
cation, such as MgCl.sub.2 or Mg(OAc).sub.2 can be added to start
the reaction. In some embodiments, the buffer may include a
crowding agent, such as PEG, Tris buffer and a potassium acetate
salt. The forward primer binding sequence is complementary or
identical to at least a portion of the blocked forward primer and
the reverse primer binding sequence is complementary or identical
to at least a portion of the blocked reverse primer. The reaction
mixture is incubated under substantially isothermal amplification
conditions, for example between 35.degree. C. and 45.degree. C. for
15 to 60 minutes, thereby amplifying the nucleic acid template.
In some embodiments, the blocked forward primer and the blocked
reverse primer comprise a 5' domain and a 3' domain separated by a
nucleotide comprising a ribobase, wherein the 5' domain is 10 to 40
nucleotides in length and the 3' domain is 11 to 25 nucleotides in
length. The forward and reverse primers, in some embodiments, bind
in opposite directions to different strands of a double-stranded
template, such that the region between the primer binding sites of
the template is amplified, as is known for pairs of amplification
primers.
In some embodiments, amplification methods and associated
compositions provided herein that include improved
ribobase-containing primers, can be used in a nucleic acid
sequencing workflow, especially a high throughput nucleic acid
sequencing workflow. In some embodiments, the reaction mixture is
formed by combining at least two different polynucleotide templates
comprising both a first primer binding sequence and a second primer
binding sequence, a recombinase, a recombinase accessory protein, a
polymerase, a first blocked universal primer, a second optionally
blocked universal primer, dNTPs, an RNase H enzyme, and a buffer.
In some embodiments, the reaction mixture is in contact with a
support having the first blocked universal primer bound thereto,
wherein the first primer binding sequence is complementary or
identical to at least a portion of the first blocked universal
primer and the second primer binding sequence is complementary or
identical to at least a portion of the second optionally blocked
universal primer.
The nucleic acid amplification methods result in the formation of
at least two substantially monoclonal nucleic acid populations by
using the polymerase to amplify each of the at least two different
polynucleotide templates onto different sites on the solid support,
within the same reaction mixture of step (a) under substantially
isothermal conditions.
In some embodiments, the two or more different nucleic acid
templates are amplified simultaneously and/or in parallel.
In some embodiments, the second optionally blocked universal primer
is in solution (e.g., soluble primers). In some embodiments, the
second optionally blocked universal primer is immobilized on the
support.
In some embodiments, the reaction mixture and the at least two
substantially monoclonal nucleic acid populations are formed in the
same single continuous liquid phase. In some embodiments, the
reaction mixture and the at least two substantially monoclonal
nucleic acid populations are formed in a water-in-oil emulsion.
In some embodiments, the at least two different polynucleotide
templates are members of a polynucleotide library, wherein each
member of the polynucleotide library comprises the first primer
binding sequence and the second primer binding sequence. In some
embodiments, nucleic acids of the at least two substantially
monoclonal nucleic acid populations are sequenced.
In some embodiments, the first blocked universal primer and the
second optionally blocked universal primer when it is present in a
blocked configuration comprise a 5' domain and a 3' domain
separated by a nucleotide comprising a ribobase, wherein the 5'
domain is 10 to 40 nucleotides in length and the 3' domain is 10 to
25 nucleotides in length. In some embodiments, embodiments, the
reaction mixture comprises a first and second blocked primer
wherein the 5' domain is 15 to 30 nucleotides in length. In some
embodiments, the 5' domain of the blocked primers is 15 to 50
nucleotides in length. In some embodiments, the reaction mixture
comprises a blocked primer wherein the 3' domain is 14 to 25
nucleotides in length. In some embodiments, the 3' domain is 15 to
25 nucleotides in length. In some embodiments, a 3' nucleotide of
the 3' domain of the forward primer is mismatched to the forward
primer binding sequence.
In some embodiments, the ribobase separating the 5' domain and the
3' domain of the blocked primer comprises rU, rG or rA. In some
embodiments, the ribobase separating the 5' domain and the 3'
domain of the blocked primer comprises rC. In some embodiments, the
3' domain of the blocked primers is 14 to 20 nucleotides in length
and the ribobase is rU, rG, rC or rA.
In some embodiments, the reaction mixture used in the methods
provided herein is a composition section provided herein. The
reaction mixture can include components such as, for example, a
recombinase accessory protein such as uvsY at concentrations
provided herein. For example, the uvsY can be present, at 20 ng/ul
to 100 ng/ul. In some embodiments, the reaction mixture comprises a
recombinase selected from the group consisting of uvsX, RecA, RadA,
RadB, Rad 51, a homologue thereof, a functional analog thereof and
a combination thereof. The UvsX protein can be present, for
example, at 50-250 ng/ul or 100-200 ng/ul. In some embodiments, the
reaction mixture comprises uvsY recombinase accessory protein and
uvsX recombinase.
In some embodiments, the reaction mixture comprises an RNase H
enzyme according to any of the teachings provided in the RNase
section herein. For example, the RNase H enzyme can be E. coli
RNase HII. In some embodiments, the RNase H enzyme is present at a
limiting or especially an excess concentration as provided herein.
Useful concentrations for such RNase H enzyme is provided elsewhere
herein.
In some embodiments, the disclosure relates generally to methods
(as well as related compositions, systems and kits) for nucleic
acid synthesis, comprising: providing at least two double stranded
nucleic acid templates in a reaction mixture; and forming at least
two substantially monoclonal nucleic acid populations by clonally
amplifying the at least two double stranded nucleic acid templates
according to any of the methods described herein.
In some embodiments, clonally amplifying optionally includes
forming a reaction mixture. The reaction mixture can include a
continuous liquid phase. In some embodiments, the continuous liquid
phase includes a single continuous aqueous phase. The liquid phase
can include two or more polynucleotide templates, which can
optionally have the same nucleotide sequence, or can have
nucleotide sequences that are different from each other. In some
embodiments, at least one of the two or more polynucleotide
templates can include at least one nucleic acid sequence that is
substantially non-identical, or substantially non-complementary, to
at least one other polynucleotide template within the reaction
mixture.
In some embodiments, the two or more different nucleic acid
templates are localized, deposited or positioned at different sites
prior to the amplifying.
In some embodiments, the two or more different nucleic acid
templates are clonally amplified in solution, optionally within a
single reaction mixture, and the resulting two or more
substantially monoclonal nucleic acid populations are then
localized, deposited or positioned at different sites following
such clonal amplification.
The different sites are optionally members of an array of sites.
The array can include a two-dimensional array of sites on a surface
(e.g., of a flowcell, electronic device, transistor chip, reaction
chamber, channel, and the like), or a three-dimensional array of
sites within a matrix or other medium (e.g., solid, semi-solid,
liquid, fluid, and the like).
In some embodiments, the two or more different nucleic acid
templates are amplified within a continuous liquid phase, typically
a continuous aqueous phase, of the same reaction mixture, thereby
producing two or more different and substantially monoclonal
populations of polynucleotides, each population being generated via
amplification of a single polynucleotide template present in the
reaction mixture.
In some embodiments, the continuous liquid phase is contained
within a single or same phase of the reaction mixture.
In some embodiments, the disclosure relates generally to methods
(as well as related compositions, systems and kits) for nucleic
acid synthesis, comprising: providing a double stranded nucleic
acid template; and forming a substantially monoclonal nucleic acid
population by amplifying the double stranded nucleic acid template.
Optionally, the amplifying includes clonally amplifying the double
stranded nucleic acid template.
In some embodiments, the amplifying includes performing at least
one round of amplification under substantially isothermal
conditions.
In some embodiments, the amplifying includes performing at least
two consecutive cycles of nucleic acid synthesis under
substantially isothermal conditions.
In some embodiments, the RPA methods can be used for template
walking. For example, the amplifying can include performing at
least one round of template-walking.
In some embodiments, the amplifying optionally includes performing
two different rounds of amplification within the sites or reaction
chambers. For example, the amplifying can include performing at
least one round of the RPA methods within the sites or reaction
chambers, and performing at least one round of template walking,
which may or may not use the RPA methods with blocked primes,
within the sites or reaction chambers, in any order or combination
of rounds. In some embodiments, at least two consecutive cycles in
any one or more of the rounds of amplification are performed under
substantially isothermal conditions. In some embodiments, at least
one of the rounds of amplification is performed under substantially
isothermal conditions.
In some embodiments, the nucleic acid template to be amplified is
double stranded, or is rendered at least partially double stranded
using appropriate procedures prior to amplification. (The template
to be amplified is referred to interchangeably herein as a nucleic
acid template or a polynucleotide template). In some embodiments,
the template is linear. Alternatively, the template can be
circular, or include a combination of linear and circular
regions.
In some embodiments, the double stranded nucleic acid template
includes a forward strand. The double stranded nucleic acid
template can further include a reverse strand. The forward strand
optionally includes a first primer binding site. The reverse strand
optionally includes a second primer binding site.
In some embodiments, the template already includes a first and/or
second primer binding site. Alternatively, the template optionally
does not originally include a primer binding site, and the
disclosed methods optionally include attaching or introducing a
primer binding site to the template prior to the amplifying. For
example, the method can optionally include ligating or otherwise
introducing an adapter containing a primer binding site to, or
into, the templates. The adapter can be ligated or otherwise
introduced to an end of a linear template, or within the body of a
linear or circular template. Optionally, the template can be
circularized after the adapter is ligated or introduced. In some
embodiments, a first adapter can be ligated or introduced at a
first end of a linear template, and a second adaptor can be ligated
or introduced at a second end of the template.
In some embodiments, the amplifying includes contacting the
partially denatured template with a first blocked primer, with a
second blocked primer, or with both a first blocked primer and a
second blocked primer, in any order or combination.
In some embodiments, the first blocked primer contains a first
primer sequence. The first blocked primer optionally includes an
extendible end (e.g., a 3'OH containing end), after cleavage by the
ribo-endonuclease to liberate the blocking group and the 3' domain
of the blocked primers. The first blocked primer can optionally be
attached to a compound (e.g., a "drag tag"), or to a support (e.g.,
a bead or a surface of the site or reaction chamber).
In some embodiments, the second blocked primer contains a second
primer sequence. The second primer optionally includes an
extendible end (e.g., a 3'OH containing end) after cleavage by the
ribo-endonuclease to liberate the blocking group and the 3' domain
of the blocked primers. The second blocked primer can optionally be
attached to a compound (e.g., a "drag tag"), or to a support (e.g.,
a bead or a surface of the site or reaction chamber).
Optionally, the first blocked primer binds to the first primer
binding site to form a first primer-template duplex. The second
blocked primer can bind to the second primer binding site to form a
second primer-template duplex.
In some embodiments, amplifying includes extending the first
blocked primer (after cleavage by the ribo-endonuclease to liberate
the blocking group and the 3' domain of the blocked primers) to
form an extended first primer. For example, amplifying can include
extending the first blocked primer of the first primer-template
duplex to form an extended first primer.
In some embodiments, amplifying includes extending the first
blocked primer (after cleavage by the ribo-endonuclease to liberate
the blocking group and the 3' domain of the blocked primers) to
form an extended first primer. For example, amplifying can include
extending the first blocked primer of the first primer-template
duplex to form an extended first primer.
In some embodiments, the amplifying includes forming a partially
denatured template. For example, the amplification can include
partially denaturing the double stranded nucleic acid template.
In some embodiments, partially denaturing includes subjecting the
double stranded nucleic acid template to partially denaturing
conditions.
In some embodiments, partially denaturing conditions include
treating or contacting the nucleic acid templates to be amplified
with one or more enzymes that are capable of partially denaturing
the nucleic acid template, optionally in a sequence-specific or
sequence-directed manner.
In some embodiments, at least one enzyme catalyzes strand invasion
and/or unwinding, optionally in a sequence-specific manner.
Optionally, the one or more enzymes include one or more enzymes
selected from the group consisting of: recombinases, topoisomerases
and helicases. In some embodiments, partially denaturing the
template can include contacting the template with a recombinase and
forming a nucleoprotein complex including the recombinase.
Optionally, the template is contacted with a recombinase in the
presence of a first blocked primer, a second blocked primer, or
both a first and second blocked primer. Partially denaturing can
include catalyzing strand exchange using the recombinase and
hybridizing the first blocked primer to the first primer binding
site (or hybridizing the second blocked primer to the second primer
binding site). In some embodiments, partially denaturing includes
performing strand exchange and hybridizing both the first blocked
primer to the first primer binding site and the second blocked
primer to the second primer binding site using the recombinase.
In some embodiments, the partially denatured template includes a
single stranded portion and a double stranded portion. In some
embodiments, the single stranded portion includes the first primer
binding site. In some embodiments, the single stranded portion
includes the second primer binding site. In some embodiments, the
single stranded portion includes both the first primer binding site
and the second primer binding site.
In some embodiments, partially denaturing the template includes
contacting the template with one or more nucleoprotein complexes.
At least one of the nucleoprotein complexes can include a
recombinase. At least one of the nucleoprotein complexes can
include a blocked primer (e.g., a first primer or a second primer,
or a primer including a sequence complementary to a corresponding
primer binding sequence in the template). In some embodiments,
partially denaturing the template can include contacting the
template with a nucleoprotein complex including a primer. Partially
denaturing can include hybridizing the blocked primer of the
nucleoprotein complex to the corresponding primer binding site in
the template, thereby forming a primer-template duplex.
In some embodiments, partially denaturing the template can include
contacting the template with a first nucleoprotein complex
including a first blocked primer. Partially denaturing can include
hybridizing the first blocked primer of the first nucleoprotein
complex to the first primer binding site of the forward strand,
thereby forming a first blocked primer-template duplex.
In some embodiments, partially denaturing the template can include
contacting the template with a second nucleoprotein complex
including a second blocked primer. Partially denaturing can include
hybridizing the second blocked primer of the second nucleoprotein
complex to the second primer binding site of the reverse strand,
thereby forming a second primer-template duplex.
In some embodiments, the disclosed methods (and related
compositions, systems and kits) can further include one or more
primer extension steps. For example, the methods can include
extending a primer via nucleotide incorporation using a polymerase.
As understood with the current RPA methods and blocked primers,
before primer extension can proceed the RNase H enzyme (e.g. RNase
HII) cleaves the primer at the ribobase location. The 5' domain
remains hybridized to the template with a 3'OH group available for
primer extension, while the 3' domain containing the blocking group
is dispersed into the reaction mixture and does not participate in
nucleic acid amplification.
In some embodiments, extending a primer includes contacting the
hybridized primer with a polymerase and one or more types of
nucleotides under nucleotide incorporation conditions. Typically,
extending a primer occurs in a template-dependent fashion.
In some embodiments, the methods (and related compositions, systems
and kits) include extending the first primer by incorporating one
or more nucleotides into the first primer of the first
primer-template duplex using the polymerase, thereby forming an
extended first primer.
In some embodiments, the methods (and related compositions, systems
and kits) include binding a second blocked primer to the second
primer binding site of the first extended primer by any suitable
method (e.g., ligation or hybridization).
In some embodiments, the methods (and related compositions, systems
and kits) include extending the second primer by incorporating one
or more nucleotides into the second primer of the second
primer-template duplex using the polymerase, thereby forming an
extended second primer. However, before primer extension can
proceed the RNase H enzyme (e.g. RNase HII) cleaves the hybridized
primer at the ribobase location. The 5' domain remains hybridized
to the template with a 3'OH group available for primer extension,
while the 3' domain containing the blocking group is dispersed into
the reaction mixture and does not participate in nucleic acid
amplification.
In some embodiments, extending the first primer results in
formation of a first extended primer. The first extended primer can
include some or all of the sequence of the reverse strand of the
template. Optionally, the first extended primer includes a second
primer binding site.
In some embodiments, extending the second primer results in
formation of a second extended primer. The second extended primer
can include some or all of the sequence of the forward strand of
the template. Optionally, the second extended primer includes a
first primer binding site.
In some embodiments, the methods are performed without subjecting
the double stranded nucleic acid template to extreme denaturing
conditions during the amplifying. For example, the methods can be
performed without subjecting the nucleic acid template(s) to
temperatures equal to or greater than the Tm of the template(s)
during the amplifying. In some embodiments, the methods can be
performed without contacting the template(s) with chemical
denaturants such as NaOH, urea, guanidium, and the like, during the
amplifying. In some embodiments, the amplifying includes
isothermally amplifying.
In some embodiments, the methods are performed without subjecting
the nucleic acid template(s) to extreme denaturing conditions
during 2, 3, 4, 5, 10, 15, 20, 25, and 30 consecutive cycles on the
low end of the range and 5, 10, 15, 20, 25, 30, or 50 consecutive
cycles on the high end of the range of nucleic acid synthesis. For
example, the methods can include 2, 3, 4, 5, 10, 15, 20, 25, and 30
consecutive cycles on the low end of the range and 5, 10, 15, 20,
25, 30, or 50 consecutive cycles on the high end of the range of
nucleic acid synthesis without contacting the nucleic acid
template(s) with a chemical denaturant or raising the temperature
above 50 or 55.degree. C. In some embodiments, the methods can
include performing 2, 3, 4, 5, 10, 15, 20, 25, and 30 consecutive
cycles on the low end of the range and 5, 10, 15, 20, 25, 30, or 50
consecutive cycles on the high end of the range of nucleic acid
synthesis without subjecting the nucleic acid template(s) to
temperatures that are greater than 25, 20, 15, 10, 5, 2 or
1.degree. C. below the actual or calculated Tm of the template, or
population of templates (or the actual or calculated average Tm of
the template, or population of templates). The consecutive cycles
of nucleic acid synthesis may or may not include intervening steps
of partial denaturation and/or primer extension.
In some embodiments, the disclosed methods (and related
compositions, systems and kits) can further include linking one or
more extended primer strands to a support. The linking can
optionally be performed during the amplifying, or alternatively
after the amplification is complete. In some embodiments, the
support includes multiple instances of a second blocked primer, and
the methods can include hybridizing at least one of the extended
first primer strands to a second blocked primer of the support.
In some embodiments, the disclosed methods (and related
compositions, systems and kits) can further include linking one or
more extended second primer strands to a support. In some
embodiments, the support is attached to a first blocked primer. For
example, the support can include multiple instances of a first
blocked primer, and the methods can include hybridizing at least
one of the extended second primers to a first blocked primer of the
support, thereby linking the extended second primer to the support.
For example, the first primer can hybridize to a first primer
binding site in the extended second primer.
In some embodiments, the support is attached to a second blocked
primer. For example, the support can include multiple instances of
a second blocked primer, and the methods can include hybridizing at
least one of the extended first primers to a second blocked primer
of the support, thereby linking the extended first primer to the
support. For example, the first blocked primer can hybridize to a
second primer binding site in the extended first primer.
In some embodiments, the support includes both at least one first
blocked primer and at least one second blocked primer, and the
disclosed methods (and related compositions, systems and kits)
including linking both an extended first primer and an extended
second primer to the support.
In some embodiments, the support is attached to a target-specific
blocked primer. The target-specific primer optionally hybridizes
(or is capable of hybridizing) to a first subset of templates
within the reaction mixture, but is unable to bind to a second
subset of templates within the reaction mixture.
In some embodiments, the support is attached to a universal blocked
primer. The universal primer optionally hybridizes (or is capable
of hybridizing) to all, or substantially all, of the templates
within the reaction mixture.
In some embodiments, the reaction mixture includes a first support
covalently attached to a first target-specific blocked primer and a
second support covalently attached to a second target-specific
blocked primer, and wherein the first and second target-specific
primers are different from each other.
In some embodiments, the first target-specific blocked primer is
substantially complementary to a first target nucleic acid sequence
and the second target-specific blocked primer is substantially
complementary to a second target nucleic acid sequence, and wherein
the first and second target nucleic acid sequences are
different.
In some embodiments, the disclosed methods include forming a first
amplicon by amplifying a first template onto a first support, and
forming a second amplicon by amplifying a second template onto a
second support, optionally within the same continuous phase of a
reaction mixture. The first amplicon is optionally linked or
attached to the first support, and the second amplicon is
optionally linked or attached to the second support.
The disclosed methods optionally comprise producing two or more
monoclonal, or substantially monoclonal, amplicons by clonally
amplifying two or more polynucleotide templates. The two or more
polynucleotide templates are optionally clonally amplified within a
continuous liquid phase of an amplification reaction mixture. The
continuous liquid phase of the amplification reaction mixture can
include a continuous aqueous phase. In some embodiments, the
amplifying includes generating at least two substantially
monoclonal populations of amplified polynucleotides, each of said
populations being formed via amplification of a single
polynucleotide template. In some embodiments, the clonally
amplifying includes at least one round of RPA. Optionally, the
clonally amplifying includes at least one round of template
walking.
In some embodiments, the amplifying optionally includes forming an
amplification reaction mixture including a continuous liquid phase.
In some embodiments, the continuous liquid phase is a single
continuous aqueous phase. The liquid phase can include two or more
polynucleotide templates, which can optionally be different from
each other. For example, the two or more polynucleotide templates
can include at least one nucleic acid sequence that is
substantially non-identical, or substantially non-complementary, to
at least one other polynucleotide template within the amplification
reaction mixture.
In some embodiments, the amplifying optionally includes forming an
amplification reaction mixture including a single continuous
aqueous phase having two or more polynucleotide templates.
Amplifying optionally includes forming two or more substantially
monoclonal nucleic acid populations by clonally amplifying the two
or more polynucleotide templates within the single aqueous phase.
Optionally, the clonally amplifying includes at least one round of
RPA. Optionally, the clonally amplifying includes at least one
round of template walking.
In some embodiments, the disclosure relates generally to methods
(and related compositions, systems and kits) for amplifying one or
more nucleic acid templates, optionally in parallel, using
partially denaturing conditions. In some embodiments, two or more
templates are amplified using such methods, optionally in array
format. Optionally, the templates are amplified in bulk in solution
prior to distribution into the array. Alternatively, the templates
are first distributed to sites in the array and then amplified in
situ at (or within) the sites of the array.
In some embodiments, the methods can include subjecting a
double-stranded nucleic acid template including a primer binding
site on at least one strand to at least one cycle of template-based
replication using a polymerase.
In some embodiments, the at least one cycle of template-based
replication includes a partial denaturation step, an annealing
step, and an extension step.
In some embodiments, the methods include amplifying the double
stranded nucleic acid template by subjecting the template to at
least two consecutive cycles of template-based replication.
In some embodiments, the methods include partially denaturing the
template. Optionally, the methods include forming a partially
denatured template including a single stranded region. The
partially denatured template can also include a double stranded
region. The single stranded region can contain the primer binding
site.
In some embodiments, the partially denaturing includes contacting
the double stranded template with a recombinase and a blocked
primer. The recombinase and primer may form part of a nucleoprotein
complex, and the partially denaturing includes contacting the
template with the complex.
In some embodiments, the methods include forming a primer-template
duplex by hybridizing a blocked primer to the primer binding site
of the single stranded region. In some embodiments, the primed
template includes a double stranded region. Optionally, the double
stranded region does not contain a primer binding site.
In some embodiments, the methods include extending the primer of
the primer-template duplex. Optionally, the methods include forming
an extended primer.
In some embodiments, different templates can be clonally amplified
onto different discrete supports (e.g., beads or particles) without
the need for compartmentalization prior to amplification. In some
embodiments, the templates are partitioned or distributed into
emulsions prior to amplifying. Optionally, the templates are
distributed into droplets forming part of a hydrophilic phase of an
emulsion having a discontinuous hydrophilic phase and a continuous
hydrophobic phase. In some embodiments, the emulsion droplets of
the hydrophilic phase also include one or more components necessary
to practice RPA and including the blocked primers and RNase H
enzyme. For example, the emulsion droplets can include a
recombinase. Optionally, the droplets include a strand-displacing
polymerase. In some embodiments, the droplets include a
support-immobilized blocked primer and/or a solution phase blocked
primer. Optionally, the primer can bind to the template, or to an
amplification product thereof. Some suitable emulsion compositions
for use with the disclosed amplification methods can be found, for
example, in U.S. Pat. Nos. 7,622,280, 7,601,499 and 7,323,305,
incorporated by reference herein in their entireties.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits, wherein the nucleic acid
amplification further include sequencing an amplified template, or
sequencing an extended primer, (e.g. an extended first primer, or
extended second primer). The sequencing can include any suitable
method of sequencing known in the art. In some embodiments, the
sequencing includes sequencing by synthesis or sequencing by
electronic detection (e.g., nanopore sequencing). In some
embodiments, sequence includes extending a template or amplified
template, or extending a sequencing primer hybridized to a template
or amplified template, via nucleotide incorporation by a
polymerase. In some embodiments, sequencing includes sequencing a
template or amplified template that is attached to a support by
contacting the template or extended primer with a sequencing
primer, a polymerase, and at least one type of nucleotide. In some
embodiments, the sequencing includes contacting the template, or
amplified template, or extended primer, with a sequencing primer, a
polymerase and with only one type of nucleotide that does not
include an extrinsic label or a chain terminating group.
In some embodiments, the template (or amplified product) can be
deposited, localized, or positioned, to a site. In some
embodiments, multiple templates/amplified templates/extended first
primers are deposited or positioned to different sites in an array
of sites. In some embodiments, the depositing, positioning or
localizing is performed prior to amplification of the template. In
some embodiments, the depositing, positioning or localizing is
performed after the amplifying. For example, amplified templates or
extended first primers can be deposited, positioned or localized to
different sites of an array.
In some embodiments, the disclosed methods result in the production
of a plurality of amplicons, at least some of which amplicons
include a clonally amplified nucleic acid population. The clonally
amplified populations produced by the methods of the disclosure can
be useful for a variety of purposes. In some embodiments, the
disclosed methods (and related compositions, systems and kits)
optionally include further analysis and/or manipulation of the
clonally amplified populations (amplicons). For example, in some
embodiments, the numbers of amplicons exhibiting certain desired
characteristics can be detected and optionally quantified.
In some embodiments, the amplifying is followed by sequencing the
amplified product. The amplified product that is sequenced can
include an amplicon comprising a substantially monoclonal nucleic
acid population. In some embodiments, the disclosed methods include
forming or positioning single members of a plurality of amplicons
to different sites. The different sites optionally form part of an
array of sites. In some embodiments, the sites in the array of
sites include wells (reaction chambers) on the surface of an isFET
array, as described further herein.
In some embodiments, methods of downstream analysis include
sequencing at least some of the plurality of amplicons in parallel.
Optionally, the multiple templates/amplified templates/extended
first primers situated at different sites of the array are
sequenced in parallel.
In some embodiments, the sequencing can include binding a
sequencing primer to the nucleic acids of at least two different
amplicons, or at least two different substantially monoclonal
populations.
In some embodiments, the sequencing can include incorporating a
nucleotide into the sequencing primer using the polymerase.
Optionally, the incorporating includes forming at least one
nucleotide incorporation byproduct, including hydrogen ions,
protons, pyrophosphate, charge transfer or heat.
Optionally, the nucleic acid to be sequenced is positioned at a
site. The site can include a reaction chamber or well. The site can
be part of an array of similar or identical sites. The array can
include a two-dimensional array of sites on a surface (e.g., of a
flowcell, electronic device, transistor chip, reaction chamber,
channel, and the like), or a three-dimensional array of sites
within a matrix or other medium (e.g., solid, semi-solid, liquid,
fluid, and the like).
In some embodiments, the site is operatively coupled to a sensor.
The method can include detecting the nucleotide incorporation using
the sensor. Optionally, the site and the sensor are located in an
array of sites coupled to sensors.
In some embodiments, the methods (and related compositions, systems
and kits) can include detecting the presence of one or more
nucleotide incorporation byproducts at a site of the array,
optionally using the FET.
In some embodiments, the methods can include detecting a pH change
occurring within the at least one reaction chamber, optionally
using the FET.
In some embodiments, the amplified nucleic acids can be further
analyzed (e.g., sequencing) at the site of distribution without
recovering and moving the amplified products to a different site or
surface for analysis (e.g., sequencing).
In some embodiments, methods of downstream analysis include
sequencing at least some of the plurality of amplicons in parallel.
Optionally, the multiple templates/amplified templates/extended
first primers situated at different sites of the array are
sequenced in parallel.
In some embodiments, the methods (and related compositions, systems
and kits) can include depositing, positioning or localizing at
least one substantially monoclonal population at a site. The site
can form part of an array of sites.
In some embodiments, at least one of the sites includes a reaction
chamber, support, particle, microparticle, sphere, bead, filter,
flowcell, well, groove, channel reservoir, gel or inner wall of a
tube.
In some embodiments the nucleic acid templates can be distributed
into the wells of an isFET array and subsequent amplification of
templates inside the wells of the array, an optional step of
downstream analysis can be performed after the amplification that
quantifies the number of sites or wells that include amplification
product. In some embodiments, the products of the nucleic acid
amplification reactions can be detected in order to count the
number of sites or wells that include an amplified template.
For example, in some embodiments the disclosure relates generally
to methods of nucleic acid analysis, comprising: providing a sample
including a first number of polynucleotides; and distributing
single polynucleotides of the sample into different sites in an
array of sites.
In some embodiments, the methods can further include forming
substantially monoclonal nucleic acid populations by amplifying the
single polynucleotides within their respective sites.
In some embodiments, the sites remain in fluid communication during
the amplifying.
In some embodiments, the amplifying includes partially denaturing
the template.
In some embodiments, the amplifying includes subjecting the
template to partially denaturing temperatures. In some embodiments,
the template includes a low-melt sequence including a primer
binding site, which is rendered single stranded when the template
is subjected to partially denaturing temperatures.
In some embodiments, the amplifying includes partially denaturing
the template.
In some embodiments, the amplifying includes contacting at least
two different templates at two different sites of the array with a
single reaction mixture for nucleic acid amplification.
In some embodiments, the reaction mixture includes a
recombinase.
In some embodiments, the reaction mixture includes at least one
primer including a "drag-tag".
In some embodiments, the amplifying includes performing at least
one amplification cycle that includes partially denaturing the
template, hybridizing a primer to the template, and extending the
primer in a template-dependent fashion. Optionally, the amplifying
includes isothermally amplifying. In some embodiments, the
amplifying is performed under substantially isothermal
conditions.
In some embodiments, the percentage of sites containing one or more
template molecules is greater than 50% and less than 100%.
In some embodiments, the disclosed methods can further include
detecting a change in ion concentration in at least one of the
sites as a result of the at least one amplification cycle.
In some embodiments, the disclosure relates generally to methods
for detection of a target nucleic acid comprising: fractionating a
sample into a plurality of sample volumes wherein more than 50% of
the fractions contain no more than 1 target nucleic acid molecule
per sample volumes; subjecting the plurality of sample volumes to
conditions for amplification, wherein the conditions include
partially denaturing conditions; detecting a change in ion
concentration in a sample volume wherein a target nucleic acid is
present; counting the number of fractions with an amplified target
nucleic acid; and determining the quantity of target nucleic acid
in the sample. The change in ion concentration may be an increase
in ion concentration or may be a decrease in ion concentration. In
some embodiments, the method may further include combining a sample
with bead. In some embodiments, the method may include loading the
sample on a substrate wherein the substrate includes at least one
well.
In some embodiments, subjecting the target nucleic acids to
partially denaturing conditions includes contacting the target
nucleic acid molecules in their respective sample volumes with a
recombinase and a polymerase under RPA conditions.
In some embodiments, subjecting the target nucleic acids to
partially denaturing conditions includes subjecting the target
nucleic acid molecules to partially denaturing temperatures.
In some embodiments, the disclosure relates generally to
compositions (and relate methods for making and using said
compositions) comprising reagents for amplifying one or more
nucleic acid templates in parallel using partially denaturing
conditions.
In some embodiments, the disclosure relates generally to methods
for clonally amplifying a population of nucleic acid templates onto
a population of supports in an amplification reaction solution,
comprising: clonally amplifying a first template onto a first
nucleic acid template onto a first support according to any of the
methods disclosed herein, and clonally amplifying a second nucleic
acid template onto a second support according to the same method,
wherein both supports are included within a single continuous
liquid phase during the amplifying.
In some embodiments, a method is provided of generating a localized
clonal population of immobilized clonal amplicons of a
single-stranded template sequence using a template-walking method,
comprising: (a) attaching the single-stranded template sequence
("template 1") to an immobilization site ("IS1"), wherein IS1
comprises multiple copies of an immobilized blocked primer ("IS1
primer") which can hybridize substantially to template 1, and
template 1 is attached to IS1 by hybridization to an IS1 primer,
and (b) amplifying template 1 using IS1 primer and a
non-immobilized optionally blocked RNase cleavable primer ("SP1
primer") in solution, wherein amplified strands that are
complementary to the single-stranded template 1 cannot hybridize
substantially when single-stranded to primers on IS1, wherein
amplification generates a localized clonal population of
immobilized clonal amplicons around the point of initial
hybridization of template 1 to IS1. In methods provided in this
section that include an IS1 and IS1 primer, a polymerase,
recombinase, and associated proteins are used to practice to
methods, along with at least one blocked RNase cleavable
primer.
Also provided is a method of generating separated and immobilized
clonal populations of a first template sequence ("template 1") and
a second template sequence ("template 2"), comprising amplifying
the first and second template sequence to generate a population of
clonal amplicons of template 1 substantially attached to first
immobilization site ("IS1") and not to a second immobilization site
("IS2"), or a population of clonal amplicons of template 2
substantially attached to IS2 and not to IS1, wherein: (a) both
templates and all amplicons are contained within the same
continuous liquid phase, where the continuous liquid phase is in
contact with a first and second immobilization site (respectively,
"IS1" and "IS2"), and where IS1 and IS2 are spatially separated,
(b) template 1 when in single-stranded form comprises a first
subsequence ("T1-FOR") at one end, and a second subsequence
("T1-REV") at its opposite end, (c) template 2 when in
single-stranded form comprises a first subsequence ("T2-FOR") at
one end, and a second subsequence ("T2-REV") at its opposite end,
(d) IS1 comprises multiple copies of an immobilized nucleic acid
optionally blocked RNase cleavable primer ("IS1 primer") that can
hybridize substantially to T1-FOR and T2-FOR when T1 and T2 are
single-stranded, (e) IS2 comprises multiple copies of an
immobilized optionally blocked RNase cleavable primer ("IS2
primer") that can hybridize substantially to both T1-FOR and T2-FOR
when T1 and T2 are single-stranded, (f) the reverse complement of
T1-REV when single-stranded cannot hybridize substantially to
optionally blocked primers on IS1, but can hybridize substantially
to a non-immobilized optionally blocked RNase cleavable primer
("SP1") in the continuous liquid phase; and (g) the reverse
complement of T2-REV when single-stranded cannot hybridize
substantially to primers on IS2, but can hybridize substantially to
a non-immobilized optionally blocked RNase cleavable primer ("SP2")
in the continuous liquid phase. At least one of the primers in the
methods disclosed in this paragraph is a blocked RNase cleavable
primer. In some embodiments, at least one of the immobilized
primers in this paragraph is a blocked RNase cleavable primer.
In some embodiments, in any method described herein, any nucleic
acid that has dissociated from one immobilization site is capable
of substantially hybridizing to both immobilization sites and any
movement (e.g., movement by diffusion, convection) of said
dissociated nucleic acid to another immobilization site is not
substantially retarded in the continuous liquid phase.
In some embodiments, in any method described herein, the continuous
liquid phase is in simultaneous contact with IS1 and IS2.
In some embodiments, in any method described herein, a first
portion of a template that is bound by an immobilized primer does
not overlap with a second portion of the template whose complement
is bound by a non-immobilized primer.
In some embodiments, in any method described herein, at least one
template to be amplified is generated from an input nucleic acid
after the nucleic acid is placed in contact with at least one
immobilization site.
In some embodiments, any method described herein comprising the
steps of: (a) contacting a support comprising immobilized primers
with a single-stranded nucleic acid template, wherein: hybridizing
a first immobilized primer to a primer-binding sequence (PBS) on
the template (b) extending the hybridized first primer in
template-dependent extension to form an extended strand that is
complementary to the template and at least partially hybridized to
the template; (c) partially denaturing the template from the
extended complementary strand such that at least a portion of the
PBS is in single-stranded form ("free portion"); (d) hybridizing
the free portion to a non-extended, immobilized second primer (e)
extending the second primer in template-dependent extension to form
an extended strand that is complementary to the template (f)
optionally, separating the annealed extended immobilized nucleic
acid strands from one another. In methods provided herein, a
polymerase, recombinase, and associated proteins are used to
practice to methods, along with at least one blocked RNase
cleavable primer.
In some embodiments, during amplification, nucleic acid duplexes
are formed comprising a starting template and/or amplified strands;
which duplexes are not subjected during amplification to conditions
that would cause complete denaturation of a substantial number of
duplexes.
In some embodiments, the single-stranded templates are produced by
taking a plurality of input double-stranded or single-stranded
nucleic acid sequences to be amplified (which sequence may be known
or unknown) and appending or creating a first universal adaptor
sequence and a second universal adaptor sequence onto the ends of
at least one input nucleic acid; wherein said first universal
adaptor sequence hybridizes to IS1 primer and/or IS2 primer, and
the reverse complement of said second universal adaptor sequence
hybridizes to at least one non-immobilized primer. The adaptors can
be double-stranded or single-stranded.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions, systems, kits and apparatuses, for
nucleic acid amplification, comprising multiplex nucleic acid
amplification, which includes amplifying within a single reaction
mixture different nucleic acid target sequences from a sample
containing a plurality of different nucleic acid target sequences,
the amplifying including generating a plurality of 2-50, or at
least fifty different amplified target sequences (or more) by
contacting at least a portion of the sample with a polymerase and a
plurality of primers under isothermal amplification conditions.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions, systems, kits and apparatuses, for
nucleic acid amplification, comprising generating substantially
monoclonal nucleic acid populations by re-amplifying the amplicons
from the multiplex nucleic acid amplification using a nucleic acid
amplification reaction (e.g., a recombinase).
Optionally, methods for multiplex nucleic acid amplification can
further include a recombinase-mediated nucleic acid amplification
method which includes re-amplifying at least some of the 2-50 or
the at least fifty different amplified target sequences by: (a)
forming a reaction mixture including a single continuous liquid
phase containing (i) a plurality of supports, (ii) at least one of
the fifty different amplified target sequences, (iii) a recombinase
and (iv) an RNase H; and (b) subjecting the reaction mixture to
amplification conditions using blocked primers according to the
invention, thereby generating a plurality of supports attached to
substantially monoclonal nucleic acid populations attached
thereto.
In some embodiments, methods for nucleic acid amplification can be
conducted in water-in-oil emulsions that provide
compartmentalization.
When conducting a nucleic acid amplification using a plurality of
polynucleotide templates, clonal amplification using traditional
amplification methods typically relies on techniques such as
compartmentalization of the reaction mixture into segregated
portions or components that are not in fluid communication with
each other in order to maintain clonality and prevent
cross-contamination of different amplified populations and to
maintain adequate yields of monoclonal amplified product. Using
such conventional amplification methods, it is typically not
feasible to clonally amplify polynucleotide templates within the
same reaction mixture without resorting to compartmentalization or
distribution of the reaction mixture into separate compartments or
vessels, because any polynucleotides within the reaction mixture
(including templates and/or amplified products) will tend to
migrate randomly through the mixture due to diffusion and/or
Brownian motion during such amplification. Such diffusion or
migration typically increases the incidence of polyclonal
amplification and thus very few, if any, monoclonal populations
will be produced.
One suitable technique to reduce the production of polyclonal
populations in conventional amplification methods uses physical
barriers to separate individual amplification reactions into
discrete compartments. For example, emulsion amplification uses
water-in-oil microreactors, where an oil phase includes many
separate, i.e., discontinuous, aqueous reaction compartments. Each
compartment serves as an independent amplification reactor, thus
the entire emulsion is capable of supporting many separate
amplification reactions in separate (discontinuous) liquid phases
in a single reaction vessel (e.g., an Eppendorf tube or a well).
Similarly, an amplification "master mix" can be prepared and
distributed into separate reaction chambers (e.g., an array of
wells), creating a set of discrete and separate phases, each of
which defines a separate amplification reaction. Such separate
phases can be further sealed off from each other prior to
amplification. Such sealing can be useful in preventing
cross-contamination between parallel and separate reactions.
Exemplary forms of sealing can include use of lids or phase
barriers (e.g., mineral oil layer on top of an aqueous reaction) to
compartmentalize the PCR reactions into individual and discrete
compartments, between which transfer of reaction components does
not occur.
Other techniques to prevent cross-contamination and reduce
polyclonality rely on immobilization of one or more reaction
components (for example, one or more templates and/or primers)
during amplification to prevent cross contamination of
amplification reaction products and consequent reduction in
monoclonality. One such example includes bridge amplification,
where all of the primers required for amplification (e.g., forward
and reverse primer) are attached to the surface of a matrix
support. In addition to such immobilization, additional
immobilization components can be included in the reaction mixture.
For example, the polynucleotide template and/or amplification
primers cam be suspended in gels or other matrices during the
amplification so as to prevent migration of amplification reaction
products from the site of synthesis. Such gels and matrices
typically require to be removed subsequently, requiring the use of
appropriate "melting" or other recovery steps and consequent loss
of yield.
In some embodiments, the disclosure provides methods for performing
substantially clonal amplification of multiple polynucleotide
templates in parallel in a single continuous liquid phase of a
reaction mixture, without need for compartmentalization or
immobilization of multiple reaction components (e.g., both primers)
during amplification. Instead, mixtures of polynucleotide templates
in solution can be directly contacted with amplification reaction
components and a suitable surface or support having a first primer
attached thereto. Other components required for amplification can
be provided in the same continuous liquid phase, including a
polymerase, one or more types of nucleotide and optionally a second
primer. In some embodiments, the reaction mixture also includes a
recombinase. Optionally, the reaction mixture further includes at
least one agent selected from the group consisting of: a diffusion
limiting agent, a sieving agent, and a crowding agent. Examples of
amplification mixtures suitable for achieving monoclonal
amplification of templates contained in a single continuous liquid
phase are described further herein. Optionally, different templates
can be amplified onto different locations on a single surface or
support, or different templates can be amplified onto different
surfaces or different supports within the same reaction
mixture.
In some embodiments, methods for nucleic acid amplification
comprise hybridization to the template of additional primers
wherein the reaction mixture comprises at least one blocked primer
of the invention. For example, a second primer can be a reverse
amplification primer which hybridizes to at least a portion of one
strand of a polynucleotide. In some embodiments, a second primer
comprises an extendible 3' end. In some embodiment, a second primer
is not attached to a surface.
In some embodiments, a third primer can be a forward amplification
primer which hybridizes to at least a portion of one strand of a
polynucleotide. In some embodiments, a third primer comprises an
extendible 3' end. In some embodiment, a third primer is not
attached to a surface. In some embodiments, a third primer
comprises a binding partner or affinity moiety (e.g., biotin) for
enriching the amplified nucleic acids.
In some embodiments, primers (e.g., first, second and third
primers) comprise single-stranded oligonucleotides.
In some embodiments, at least a portion of a primer can hybridize
with a portion of at least one strand of a polynucleotide in the
reaction mixture. For example, at least a portion of a primer can
hybridize with a nucleic acid adaptor that is joined to one or both
ends of the polynucleotide. In some embodiments, at least a portion
of a primer can be partially or fully complementary to a portion of
the polynucleotide or to the nucleic acid adaptor. In some
embodiments, the nucleic acid adaptor includes one or more
universal sequences, for example universal primer binding
sequences. In some embodiments, a primer can be compatible for use
in any type of sequencing platform including chemical degradation,
chain-termination, sequence-by-synthesis, pyrophosphate, massively
parallel, ion-sensitive, and single molecule platforms.
In some embodiments, a primer (e.g., first, second or third primer)
can have a 5' or 3' overhang tail (tailed primer) that does not
hybridize with a portion of at least one strand of a polynucleotide
in the reaction mixture. Typically, the blocked primers do not have
an overhang tail, however when the reaction mixture comprises an
additional primer that is a standard (non-blocked primer) it may
comprise an overhang tail. In some embodiments, a tailed primer can
be any length, including 1-50 or more nucleotides in length.
In some embodiments, nucleic acids that have been amplified
according to the present teachings can be used in any nucleic acid
sequencing workflow, including sequencing by oligonucleotide probe
ligation and detection (e.g., SOLiD.TM. from Life Technologies, WO
2006/084131), probe-anchor ligation sequencing (e.g., Complete
Genomics.TM. or Polonator.TM.), sequencing-by-synthesis (e.g.,
Genetic Analyzer and HiSeq.TM., from Illumina), pyrophosphate
sequencing (e.g., Genome Sequencer FLX from 454 Life Sciences),
ion-sensitive sequencing (e.g., Personal Genome Machine (PGM.TM.)
and Ion Proton.TM. Sequencer, both from Ion Torrent Systems, Inc.),
single molecule sequencing platforms (e.g., HeliScope.TM. from
Helicos.TM.) and nanopore sequencing via read of individual bases
as they pass through the nanopores (e.g. MinION from Oxford
Nanopore Technologies).
In some embodiments, nucleic acid that have been amplified
according to the present teachings can be sequenced by any
sequencing method, including sequencing-by-synthesis, ion-based
sequencing involving the detection of sequencing byproducts using
field effect transistors (e.g., FETs and ISFETs), chemical
degradation sequencing, ligation-based sequencing, hybridization
sequencing, pyrophosphate detection sequencing, capillary
electrophoresis, gel electrophoresis, next-generation, massively
parallel sequencing platforms, sequencing platforms that detect
hydrogen ions or other sequencing by-products, and single molecule
sequencing platforms. In some embodiments, a sequencing reaction
can be conducted using at least one sequencing primer that can
hybridize to any portion of the polynucleotide constructs,
including a nucleic acid adaptor or a target polynucleotide.
In some embodiments, the sequencing can be conducted on a support
having a plurality of sequencing reaction sites arranged in an
array on the support, where the sequencing reaction sites are
capacitively coupled to at least one sensor that detects the
presence or a change in concentration of a nucleotide incorporation
byproduct (e.g., pyrophosphate, hydrogen ion, charge transfer,
heat). In some embodiments, the support includes at least 10.sup.2,
10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9 reaction sites, where each site is capacitively coupled to
at least one sensor. In some embodiments, the sensor comprises a
field effect transistor, including those described in in U.S. Pat.
No. 7,948,015 to Rothberg et al.; and Rothberg et al, U.S. Patent
Publication No. 2009/0026082, hereby incorporated by reference in
their entireties. Other examples of methods of detecting
polymerase-based extension can be found, for example, in Pourmand
et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006); Purushothaman
et al., IEEE ISCAS, IV-169-172; Anderson et al, Sensors and
Actuators B Chem., 129: 79-86 (2008); Sakata et al., Angew. Chem.
118:2283-2286 (2006); Esfandyapour et al., U.S. Patent Publication
No. 2008/01666727; and Sakurai et al., Anal. Chem. 64: 1996-1997
(1992). In addition detection may be based on a change in
capacitance, impedance or conductivity or voltammetry.
In various exemplary embodiments, the methods, systems, and
computer readable media described herein may advantageously be used
to process and/or analyze data and signals obtained from electronic
or charged-based nucleic acid sequencing. In electronic or
charged-based sequencing (such as, pH-based sequencing), a
nucleotide incorporation event may be determined by detecting ions
(e.g., hydrogen ions) that are generated as natural by-products of
polymerase-catalyzed nucleotide extension reactions. This may be
used to sequence a sample or template nucleic acid, which may be a
fragment of a nucleic acid sequence of interest, for example, and
which may be directly or indirectly attached as a clonal population
to a solid support, such as a particle, microparticle, bead, etc.
The sample or template nucleic acid may be operably associated to a
primer and polymerase and may be subjected to repeated cycles or
"flows" of nucleotide addition (which may be referred to herein as
"nucleotide flows" from which nucleotide incorporations may result)
and washing. The primer may be annealed to the sample or template
so that the primer's 3' end can be extended by a polymerase
whenever nucleotides complementary to the next base in the template
are added. Then, based on the known sequence of nucleotide flows
and on measured output signals of the chemical sensors indicative
of ion concentration during each nucleotide flow, the identity of
the type, sequence and number of nucleotide(s) associated with a
sample nucleic acid present in a reaction region coupled to a
chemical sensor can be determined.
In a typical embodiment of ion-based nucleic acid sequencing,
nucleotide incorporations can be detected by detecting the presence
and/or concentration of hydrogen ions generated by
polymerase-catalyzed extension reactions. In one embodiment,
templates, optionally pre-bound to a sequencing primer and/or a
polymerase, can be loaded into reaction chambers (such as the
microwells disclosed in Rothberg et al, cited herein), after which
repeated cycles of nucleotide addition and washing can be carried
out. In some embodiments, such templates can be attached as clonal
populations to a solid support, such as particles, bead, or the
like, and said clonal populations are loaded into reaction
chambers.
In another embodiment, the templates, optionally bound to a
polymerase, are distributed, deposited or positioned to different
sites of the array. The site of the array includes primers and the
methods can include hybridizing different templates to the primers
within different sites.
In each addition step of the cycle, the polymerase can extend the
primer by incorporating added nucleotide only if the next base in
the template is the complement of the added nucleotide. If there is
one complementary base, there is one incorporation, if two, there
are two incorporations, if three, there are three incorporations,
and so on. With each such incorporation there is a hydrogen ion
released, and collectively a population of templates releasing
hydrogen ions changes the local pH of the reaction chamber. The
production of hydrogen ions is monotonically related to the number
of contiguous complementary bases in the template (as well as the
total number of template molecules with primer and polymerase that
participate in an extension reaction). Thus, when there are a
number of contiguous identical complementary bases in the template
(i.e. a homopolymer region), the number of hydrogen ions generated,
and therefore the magnitude of the local pH change, can be
proportional to the number of contiguous identical complementary
bases. If the next base in the template is not complementary to the
added nucleotide, then no incorporation occurs and no hydrogen ion
is released. In some embodiments, after each step of adding a
nucleotide, an additional step can be performed, in which an
unbuffered wash solution at a predetermined pH is used to remove
the nucleotide of the previous step in order to prevent
misincorporations in later cycles. In some embodiments, the after
each step of adding a nucleotide, an additional step can be
performed wherein the reaction chambers are treated with a
nucleotide-destroying agent, such as apyrase, to eliminate any
residual nucleotides remaining in the chamber, which may result in
spurious extensions in subsequent cycles.
In one exemplary embodiment, different kinds of nucleotides are
added sequentially to the reaction chambers, so that each reaction
can be exposed to the different nucleotides one at a time. For
example, nucleotides can be added in the following sequence: dATP,
dCTP, dGTP, dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each
exposure followed by a wash step. The cycles may be repeated for 50
times, 100 times, 200 times, 300 times, 400 times, 500 times, 750
times, or more, depending on the length of sequence information
desired.
In some embodiments, sequencing can be performed according to the
user protocols supplied with the PGM.TM. or Proton.TM. sequencer.
Example 3 provides one exemplary protocol for ion-based sequencing
using the Ion Torrent PGM.TM. sequencer (Ion Torrent.TM. Systems,
Thermo Fisher Scientific, CA). In some embodiments, sequencing can
be performed according to the user protocols supplied with the Ion
S5 or Ion S5 XL sequencer (Ion Torrent.TM. Systems).
In some embodiments, the disclosure relates generally to methods
for sequencing a population of nucleic acid templates, comprising:
(a) generating a plurality of amplicons by clonally amplifying a
plurality of nucleic acid templates onto a plurality of surfaces,
wherein the amplifying is performed within a single continuous
phase of a reaction mixture and wherein at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or 95% of the resulting amplicons are
substantially monoclonal or monoclonal in nature. A sufficient
number of substantially monoclonal or monoclonal amplicons can be
produced in a single amplification reaction to generate at least
100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1 GB or 2 GB of
AQ20 sequencing reads on an Ion Torrent PGM.TM. 314, 316 or 318
sequencer. With respect to related high throughput systems, a
sufficient number of substantially monoclonal or monoclonal
amplicons can be produced in a single amplification reaction to
generate at least 100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1
GB, 2 GB, 5 GB, 10 GB or 15 GB of AQ20 sequencing reads on an Ion
Torrent Proton, S5 or S5XL sequencer. The term "AQ20 and its
variants, as used herein, refers to a particular method of
measuring sequencing accuracy in the Ion Torrent PGM.TM. sequencer.
Accuracy can be measured in terms of the Phred-like Q score, which
measures accuracy on logarithmic scale that: Q10=90%, Q20=99%,
Q30=99.9%, Q40=99.99%, and Q50=99.999%. For example, in a
particular sequencing reaction, accuracy metrics can be calculated
either through prediction algorithms or through actual alignment to
a known reference genome. Predicted quality scores ("Q scores") can
be derived from algorithms that look at the inherent properties of
the input signal and make fairly accurate estimates regarding if a
given single base included in the sequencing "read" will align. In
some embodiments, such predicted quality scores can be useful to
filter and remove lower quality reads prior to downstream
alignment. In some embodiments, the accuracy can be reported in
terms of a Phred-like Q score that measures accuracy on logarithmic
scale such that: Q10=90%, Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99%,
and Q50=99.999%. In some embodiments, the data obtained from a
given polymerase reaction can be filtered to measure only
polymerase reads measuring "N" nucleotides or longer and having a Q
score that passes a certain threshold, e.g., Q10, Q17, Q100
(referred to herein as the "NQ17" score). For example, the 100Q20
score can indicate the number of reads obtained from a given
reaction that are at least 100 nucleotides in length and have Q
scores of Q20 (99%) or greater. Similarly, the 200Q20 score can
indicate the number of reads that are at least 200 nucleotides in
length and have Q scores of Q20 (99%) or greater.
In some embodiments, the accuracy can also be calculated based on
proper alignment using a reference genomic sequence, referred to
herein as the "raw" accuracy. This is single pass accuracy,
involving measurement of the "true" per base error associated with
a single read, as opposed to consensus accuracy, which measures the
error rate from the consensus sequence which is the result of
multiple reads. Raw accuracy measurements can be reported in terms
of "AQ" scores (for aligned quality). In some embodiments, the data
obtained from a given polymerase reaction can be filtered to
measure only polymerase reads measuring "N" nucleotides or longer
having a AQ score that passes a certain threshold, e.g., AQ10,
AQ17, AQ100 (referred to herein as the "NAQ17" score). For example,
the 100AQ20 score can indicate the number of reads obtained from a
given polymerase reaction that are at least 100 nucleotides in
length and have AQ scores of AQ20 (99%) or greater. Similarly, the
200AQ20 score can indicate the number of reads that are at least
200 nucleotides in length and have AQ scores of AQ20 (99%) or
greater.
In some embodiments, the present teachings provide systems for
nucleic acid amplification, comprising any combination of: beads
attached with a plurality of at least one blocked primer of the
invention (first primer, second primer, third primer,)
polynucleotides, recombinase, recombinase loading protein,
single-stranded binding protein (SSB), polymerase, nucleotides,
ATP, RNase H enzyme, phosphocreatine, creatine kinase,
hybridization solutions, and/or washing solutions. A system can
include all or some of these components. In some embodiments,
systems for nucleic acid amplification can further comprise any
combination of: buffers and/or cations (e.g., divalent
cations).
In some embodiments, the present teachings provide kits for nucleic
acid amplification. In some embodiments, kits include any reagent
that can be used for nucleic acid amplification. In some
embodiments, kits include any combination of: beads attached with a
plurality of at least one blocked primer of the invention (first
primer, second primer, third primer), polynucleotides, recombinase,
recombinase loading protein, single-stranded binding protein (SSB),
polymerase, nucleotides, ATP, RNase H enzyme, phosphocreatine,
creatine kinase, hybridization solutions, washing solutions,
buffers and/or cations (e.g., divalent cations). A kit can include
all or some of these components.
In some embodiments, the disclosure relates generally to methods,
compositions, systems useful for amplifying different nucleic acid
templates in parallel in a plurality of compartmentalized reaction
volumes, as opposed to amplification within a single continuous
liquid phase. For example, the nucleic acid templates can be
distributed or deposited into an array of reaction chambers, or an
array of reaction volumes, such that at least two such chambers or
volumes in the array each receive a single nucleic acid template.
In some embodiments, a plurality of separate reaction volumes is
formed. The reaction chambers (or reaction volumes) can optionally
be sealed prior to amplification. In another embodiment, the
reaction mixture can be compartmentalized or separated into a
plurality of microreactors dispersed within a continuous phase of
an emulsion, the compartmentalized or separate reaction volumes
optionally do not mix or communicate, or are not capable of mixing
or communicating, with each other. In some embodiments, at least
some of the reaction chambers (or reaction volumes) include a
recombinase, and optionally a polymerase. The polymerase can be a
strand-displacing polymerase.
In some embodiments, the disclosure relates generally to
compositions, systems, methods, apparatuses and kits for nucleic
acid synthesis and/or amplification including emulsions. As used
herein, the term "emulsion" includes any composition including a
mixture of a first liquid and a second liquid, wherein the first
and second liquids are substantially immiscible with each other.
Typically, one of the liquids is hydrophilic while the other liquid
is hydrophobic. Typically, the emulsion includes a dispersed phase
and a continuous phase. For example, the first liquid can form a
dispersed phase that is dispersed in the second liquid, which forms
the continuous phase. The dispersed phase is optionally comprised
predominantly of the first liquid. The continuous phase is
optionally comprised predominantly of the second liquid. In various
embodiments, the same two liquids can form different types of
emulsions. For example, in a mixture including both oil and water
can form, firstly, an oil-in-water emulsion, where the oil is the
dispersed phase, and water is the dispersion medium. Secondly, they
can form a water-in-oil emulsion, where water is the dispersed
phase and oil is the external phase. Multiple emulsions are also
possible, including a "water-in-oil-in-water" emulsion and an
"oil-in-water-in-oil" emulsion. In some embodiments, the dispersed
phase includes one or more microreactors in which nucleic acid
templates can be individually amplified. One or more microreactors
can form compartmentalized reaction volumes in which separate
amplification reactions can occur. One example of a suitable
vehicle for nucleic acid amplification includes a water-in-oil
emulsion wherein the water-based phase includes several aqueous
microreactors that are dispersed within an oil phase of an
emulsion. In some embodiments, the emulsion can further include an
emulsifier or surfactant. The emulsifier or surfactant can be
useful in stabilizing the emulsion under nucleic acid synthesis
conditions.
In some embodiments, the disclosure relates generally to a
composition comprises an emulsion including a reaction mixture. The
emulsion can include an aqueous phase. The aqueous phase can be
dispersed in a continuous phase of the emulsion. The aqueous phase
can include one or more microreactors. In some embodiments, the
reaction mixture is contained in a plurality of liquid phase
microreactors within a phase of an emulsion. Optionally, the
reaction mixture includes a recombinase. Optionally, the reaction
mixture includes a plurality of different polynucleotides.
Optionally, the reaction mixture includes a plurality of supports.
Optionally, the reaction mixture includes any combination of a
recombinase, a plurality of different polynucleotides and/or a
plurality of supports. Optionally, at least one of the supports can
be attached to a substantially monoclonal nucleic acid
population.
In some embodiments, the disclosure relates generally to a
composition comprising a reaction mixture, the reaction mixture
including (i) a plurality of supports, (ii) a plurality of
different polynucleotides and (iii) a recombinase, the reaction
mixture contained in a plurality of liquid phase microreactors in
an emulsion.
In some embodiments, the disclosure relates generally to a
composition comprising a reaction mixture, the reaction mixture
including (i) a recombinase and (ii) a plurality of supports, at
least one of the supports being attached to a substantially
monoclonal nucleic acid population, wherein the reaction mixture is
contained in a plurality of liquid phase microreactors in an
emulsion.
In some embodiments, the disclosure relates generally to a
composition comprising an emulsion. Optionally, the emulsion
comprises a hydrophilic phase and a hydrophobic phase. Optionally,
the emulsion comprises a hydrophilic phase dispersed in a
hydrophobic phase. Optionally, the hydrophilic phase can include
any combination of a plurality of polynucleotide templates, a
plurality of supports and/or a recombinase. Optionally, the
hydrophilic phase can include a plurality of polynucleotide
templates. Optionally, the hydrophilic phase can include a
plurality of supports. Optionally, the hydrophilic phase can
include a recombinase.
In some embodiments, a composition comprises an emulsion comprising
a hydrophilic phase and a hydrophobic phase, wherein the
hydrophilic phase includes a plurality of polynucleotide templates,
a plurality of supports and a recombinase.
In some embodiments, the disclosure relates generally to a
composition comprises an emulsion including a hydrophilic phase
dispersed in a hydrophobic phase. Optionally, the hydrophilic phase
includes a plurality of microreactors. Optionally, at least two
microreactors of the plurality includes a different polynucleotide
template. Optionally, the sequences of the different polynucleotide
templates is the same or different. Optionally, a first
microreactor includes a first polynucleotide template and a second
microreactor includes a second polynucleotide template. Optionally,
the first and the second polynucleotide templates comprise the same
or different sequences. Optionally, at least two microreactors of
the plurality includes a recombinase.
In some embodiments, a composition comprises an emulsion including
a hydrophilic phase dispersed in a hydrophobic phase, wherein the
hydrophilic phase including a plurality of microreactors, at least
two microreactors of the plurality including a different
polynucleotide template and a recombinase.
In some embodiments, the hydrophilic phase includes a plurality of
aqueous microreactors, at least two of the microreactors each
including a different polynucleotide template, a support, and a
recombinase.
Optionally, a first microreactor includes a first polynucleotide
template and a second microreactor includes a second polynucleotide
template. Optionally, the first and the second polynucleotide
templates comprise the same or different sequences.
Optionally, at least one of the plurality of supports is linked to
a plurality of first primers (e.g., forward amplification
primers).
Optionally, the reaction mixture further includes a plurality of a
second primer (e.g., reverse amplification primers).
In some embodiments, at least one of the plurality of supports
further includes a plurality of second primers.
In some embodiments, at least one of the plurality of supports
includes a plurality of first and second primers.
In some embodiments, the first and second primers comprise the same
sequences.
In some embodiments, the first and second primers comprise
different sequences. In some embodiments, the hydrophilic phase
further includes a polymerase.
In some embodiments, the polymerase comprises a strand displacing
polymerase. In some embodiments, the hydrophilic phase includes
nucleotides.
In some embodiments, the disclosure relates generally to methods
(as well as associated compositions and systems) for nucleic acid
synthesis, comprising: (a) forming a reaction mixture; and (b)
subjecting the reaction mixture to amplification conditions.
Optionally, the reaction mixture is contained within a hydrophilic
phase of an emulsion. Optionally, the emulsion includes a
hydrophilic phase and a hydrophobic phase. Optionally, the emulsion
comprises a hydrophilic phase dispersed in a hydrophobic phase.
Optionally, the reaction mixture contains any combination of a
plurality of supports, a plurality of different polynucleotides
and/or a recombinase. Optionally, the reaction mixture contains a
plurality of supports. Optionally, the reaction mixture contains a
plurality of different polynucleotides. Optionally, the sequences
of the different polynucleotide templates is the same or different.
Optionally, a first microreactor includes a first polynucleotide
template and a second microreactor includes a second polynucleotide
template. Optionally, the first and the second polynucleotide
templates comprise the same or different sequences. Optionally, the
reaction mixture contains a recombinase. Optionally, the
amplification conditions include isothermal or thermo-cycling
temperature conditions. Optionally, the method further includes
forming at least two supports subjecting the emulsion to
amplification conditions results in forming a plurality of
supports, wherein at least two of the supports are each
independently attached to a substantially monoclonal nucleic acid
population.
In some embodiments, the disclosure relates generally to methods
(as well as associated compositions and systems) for nucleic acid
synthesis, comprising: (a) forming a reaction mixture containing a
plurality of supports, a plurality of different polynucleotides and
a recombinase, the reaction mixture contained within a hydrophilic
phase of an emulsion; and (b) subjecting the emulsion including the
reaction mixture to isothermal amplification conditions, thereby
generating a plurality of supports and a substantially monoclonal
nucleic acid population attached thereto.
In some embodiments, the emulsion includes a water-in-oil emulsion.
In some embodiments, the liquid phase microreactors comprise a
hydrophilic phase. In some embodiments, the emulsion comprises a
hydrophilic phase dispersed in a hydrophobic phase. In some
embodiments, the reaction mixture is formed in a single reaction
vessel. Optionally, the sequences of the plurality of different
polynucleotide templates is the same or different. Optionally, a
first polynucleotide template includes a first sequence and a
second polynucleotide template includes a second sequence.
Optionally, the first and the second polynucleotide template
sequences are the same or different. Optionally, at least one of
the plurality of supports is linked to a plurality of first primers
(e.g., forward amplification primers). Optionally, the reaction
mixture further includes a plurality of a second primer (e.g.,
reverse amplification primers). In some embodiments, at least one
of the plurality of supports further includes a plurality of second
primers. In some embodiments, at least one of the plurality of
supports includes a plurality of first and second primers. In some
embodiments, the first and second primers comprise the same
sequences. In some embodiments, the first and second primers
comprise different sequences. In some embodiments, the nucleic acid
synthesis method further includes recovering from the reaction
mixture at least some of the supports attached to substantially
nucleic acid monoclonal populations. In some embodiments, the
nucleic acid synthesis method further includes depositing onto a
surface at least some of the supports attached to the substantially
monoclonal nucleic acid populations. In some embodiments, the
nucleic acid synthesis method further includes forming an array by
depositing onto a surface at least some of the supports attached to
the substantially monoclonal nucleic acid populations. In some
embodiments, the nucleic acid synthesis method further includes
sequencing at least one substantially monoclonal nucleic acid
population attached to the support. In some embodiments, the
support comprises a bead, particle, a planar surface, or an
interior wall of a channel or tube. In some embodiments, the
reaction mixture further includes a polymerase and a plurality of
nucleotides. In some embodiments, the polymerase comprises a strand
displacing polymerase.
In some embodiments, methods for nucleic acid synthesis comprise
forming an emulsion. Optionally, the emulsion comprises a
hydrophilic phase and a hydrophobic phase. Optionally, the emulsion
comprises a hydrophilic phase dispersed in a hydrophobic phase.
Optionally, the hydrophilic phase includes a plurality of
microreactors. Optionally, at least two microreactors of the
plurality include individual polynucleotide templates. Optionally,
at least two microreactors of the plurality include a different
polynucleotide template. Optionally, a first microreactor includes
a first polynucleotide template, and a second microreactor includes
a second polynucleotide template. Optionally, the first and the
second polynucleotide templates have the same or different
sequences. Optionally, at least two microreactors of the plurality
including a recombinase.
In some embodiments, the disclosure relates generally to methods
(as well as associated compositions and systems) for nucleic acid
synthesis, comprising: forming an emulsion including a hydrophilic
phase dispersed in a hydrophobic phase, the hydrophilic phase
including a plurality of microreactors, at least two microreactors
of the plurality including a different polynucleotide template and
a recombinase.
In some embodiments, the emulsion includes a water-in-oil emulsion.
In some embodiments, the hydrophilic phase further includes a
polymerase. In some embodiments, the polymerase is a strand
displacing polymerase. In some embodiments, the hydrophilic phase
includes nucleotides. In some embodiments, the emulsion is formed
in a single reaction vessel. Optionally, the sequences of the
different polynucleotide templates are the same or different.
Optionally, a first microreactor includes a first polynucleotide
template and a second microreactor includes a second polynucleotide
template. Optionally, the first and the second polynucleotide
templates comprise the same or different sequences. In some
embodiments, the at least two microreactors of the plurality
include a plurality of supports. Optionally, at least one of the
plurality of supports is linked to a plurality of first primers
(e.g., forward amplification primers). Optionally, the reaction
mixture further includes a plurality of a second primer (e.g.,
reverse amplification primers). In some embodiments, at least one
of the plurality of supports further includes a plurality of second
primers. In some embodiments, at least one of the plurality of
supports includes a plurality of first and second primers. In some
embodiments, the first and second primers comprise the same
sequences. In some embodiments, the first and second primers
comprise different sequences. In some embodiments, the hydrophilic
phase includes a reaction mixture. In some embodiments, the
reaction mixture comprises a plurality of polynucleotide templates,
a plurality of supports and a recombinase. In some embodiments,
methods for nucleic acid synthesis further comprise subjecting the
emulsion (e.g., including the reaction mixture) to isothermal
amplification conditions, thereby generating a plurality of
substantially monoclonal nucleic acid populations. In some
embodiments, the plurality of substantially monoclonal nucleic acid
populations is attached to the plurality of supports. In some
embodiments, the nucleic acid synthesis method further includes
recovering from the reaction mixture at least some of the supports
attached to substantially nucleic acid monoclonal populations. In
some embodiments, the nucleic acid synthesis method further
includes depositing onto a surface at least some of the supports
attached to the substantially monoclonal nucleic acid populations.
In some embodiments, the nucleic acid synthesis method further
includes forming an array by depositing onto a surface at least
some of the supports attached to the substantially monoclonal
nucleic acid populations. In some embodiments, the nucleic acid
synthesis method further includes sequencing at least one
substantially monoclonal nucleic acid population attached to the
support. In some embodiments, the support comprises a bead,
particle, a planar surface, or an interior wall of a channel or
tube. In some embodiments, the reaction mixture further includes a
polymerase and a plurality of nucleotides. In some embodiments, the
polymerase comprises a strand displacing polymerase.
The section headings used herein are for organizational purposes
only and are not to be construed as limiting the described subject
matter in any way.
All literature and similar materials cited in this application,
including but not limited to, patents, patent applications,
articles, books, treatises, and internet web pages are expressly
incorporated by reference in their entirety for any purpose. When
definitions of terms in incorporated references appear to differ
from the definitions provided in the present teachings, the
definition provided in the present teachings shall control.
It will be appreciated that there is an implied "about" prior to
the temperatures, concentrations, times, etc., discussed in the
present teachings, such that slight and insubstantial deviations
are within the scope of the present teachings herein.
Unless otherwise required by context, singular terms shall include
pluralities and plural terms shall include the singular.
The use of "comprise", "comprises", "comprising", "contain",
"contains", "containing", "include", "includes", and "including"
are not intended to be limiting.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention.
Unless otherwise defined, scientific and technical terms used in
connection with the present teachings described herein shall have
the meanings that are commonly understood by those of ordinary
skill in the art. Generally, nomenclatures utilized in connection
with, and techniques of, cell and tissue culture, molecular
biology, and protein and oligo- or polynucleotide chemistry and
hybridization described herein are those well-known and commonly
used in the art. Standard techniques are used, for example, for
nucleic acid purification and preparation, chemical analysis,
recombinant nucleic acid, and oligonucleotide synthesis. Enzymatic
reactions and purification techniques are performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The techniques and procedures described
herein are generally performed according to conventional methods
well known in the art and as described in various general and more
specific references that are cited and discussed throughout the
instant specification. See, e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. 2000). The nomenclatures
utilized in connection with, and the laboratory procedures and
techniques described herein are those well-known and commonly used
in the art.
As utilized in accordance with exemplary embodiments provided
herein, the following terms, unless otherwise indicated, shall be
understood to have the following meanings:
The term "monoclonal" and its variants, when used in reference to
one or more polynucleotide populations, refers to a population of
polynucleotides where at least 90% of the members of the population
share at least 90% identity at the nucleotide sequence level. As
used herein, the phrase "substantially monoclonal" and its
variants, when used in reference to one or more polynucleotide
populations, refer to one or more polynucleotide populations
wherein an amplified template polynucleotide molecule is the single
largest polynucleotide in the population. Accordingly, all members
of a monoclonal or substantially monoclonal population need not be
completely identical or complementary to each other. For example,
different portions of a polynucleotide template can become
amplified or replicated to produce the members of the resulting
monoclonal population; similarly, a certain number of "errors"
and/or incomplete extensions may occur during amplification of the
original template, thereby generating a monoclonal or substantially
monoclonal population whose individual members can exhibit sequence
variability amongst themselves. In some embodiments, a low or
insubstantial level of mixing of non-homologous polynucleotides may
occur during nucleic acid amplification reactions disclosed herein,
and thus a substantially monoclonal population may contain a
minority of diverse polynucleotides (e.g., less than 30%, less than
20%, less than 10%, less than 5%, less than 1%, less than 0.5%,
less than 0.1%, or less than 0.001%, of diverse polynucleotides).
In certain examples, at least 90% of the polynucleotides in the
population are at least 90% identical to the original single
template used as a basis for clonal amplification to produce the
substantially monoclonal population. In some embodiments, methods
for clonally amplifying provided herein, yield a population of
polynucleotides wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the members
of a population of polynucleotides share at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the template
nucleic acid from which the population was generated. In some
embodiments, methods for clonally amplifying provided herein, yield
a population of polynucleotides in which a large enough fraction of
the polynucleotides share enough sequence identity to allow
sequencing of at least a portion of the amplified template using a
high throughput sequencing system.
In some embodiments, at least 50%, 60%, 70%, 75%, 80%, 90%, 95%, or
99%, of the members of the amplicon will share greater than 90%,
95%, 97%, 99%, or 100% identity with the polynucleotide template.
In some embodiments, members of a nucleic acid population produced
using methods provided herein, can hybridize to each other under
high stringency hybridization conditions.
In some embodiments, methods provided herein generate a population
of polynucleotides that includes sufficiently few polyclonal
contaminants to be successfully sequenced in a high throughput
sequencing method. For example, methods provided herein can
generate a population of polynucleotides that produces a signal
(e.g., a sequencing signal, a nucleotide incorporation signal and
the like) that can be detected using a particular sequencing
system. Optionally, the signal can subsequently be analyzed to
correctly determine the sequence and/or base identity of any one or
more nucleotides present within any polynucleotide of the
population. Examples of suitable sequencing systems for detection
and/or analysis of such signals include the Ion Torrent sequencing
systems, such as the Ion Torrent PGM.TM. sequence systems,
including the 314, 316 and 318 systems, the Ion Torrent Proton.TM.
sequencing systems, including Proton I, (Life Technologies,
Carlsbad, Calif.) and the Ion Torrent Proton.TM. sequencing
systems, including Ion S5 and S5XL (Thermo Fisher Scientific, CA).
In some embodiments, the monoclonal amplicon permits the accurate
sequencing of at least 5 contiguous nucleotide residues on an Ion
Torrent sequencing system.
As used herein, the term "clonal amplification" and its variants
refer to any process whereby a substantially monoclonal
polynucleotide population is produced via amplification of a
polynucleotide template. In some embodiments of clonal
amplification, two or more polynucleotide templates are amplified
to produce at least two substantially monoclonal polynucleotide
populations.
As used herein, a "blocked primer" or a "3'-blocked primer" cannot
be extended by a polymerase. Typically, a 3'-OH is missing or a
chemical moiety is used to block polymerase extension. For example,
the primer may have 3'-phosphate, 3' biotin, 3' amine, C3 spacer
(3' Propyl), Spacer 9/18 either at the 3' end or close to it,
1',2'-Dideoxyribose (dSpacer), 3' Hexanediol, 2'-3'-Dideoxy,
3'-deoxy bases, inverted dT (see modified bases and spacers by
IDT), 3'-amine or any other moiety that disables polymerase
extension, see for example Table 2 in Lin-Ling et al. "Single-base
discrimination mediated by proofreading inert allele specific
primers", J. Biochem Mol. Biol. 2005 Jan. 31; 38(1):24-7.
As used herein, a "ribobase" means one or more nucleotides that are
cleavable by an RNase H enzyme. The ribobase can be an rU, rA, rC,
or rG, as non-limiting examples.
In some embodiments, the disclosure relates generally to methods,
as well as related compositions and kits, for nucleic acid
amplification, which includes cleaving with an endonuclease an
oligonucleotide that is hybridized to a template nucleic acid.
Cleaving the oligonucleotide can, optionally, be followed by an
isothermal amplification reaction, especially recombinase-mediated
amplification reactions such as RPA (recombinase-polymerase
amplification). The methods and compositions use an oligonucleotide
including a cleavable moiety, wherein the oligonucleotide is not
extendable by a polymerase. That oligonucleotide is also referred
to herein as a "blocked primer", wherein the cleavable moiety
separates a 5' domain and 3' domain of the primer, and wherein an
endonuclease, such as RNase H as disclosed herein, cleaves the
primer at the cleavable moiety location removing the block. The 5'
domain of the oligonucleotide remains hybridized to the template
nucleic acid after the block is removed. Accordingly, the
endonuclease cleaves or hydrolyzes a location or residue on the
oligonucleotide when it is hybridized to a nucleic acid template.
In some embodiments, the oligonucleotide and template nucleic acid
form a DNA:DNA duplex. In further embodiments, the endonuclease
does not cleave the oligonucleotide or blocked primer at other
nucleotide positions besides the cleavable moiety.
In some embodiments, the cleavable moiety is one or more
nucleotides that are cleavable by an RNase H enzyme, wherein the
endonuclease is RNase H. Nucleotides cleavable by RNase H include
ribobases rU, rA, rC, and rG, any of which may be present as a
single ribobase or as two or more at the desired cleavage location
in the oligonucleotide or blocked primer. In some embodiments, the
RNase H enzyme is any RNase H enzyme disclosed herein, and is
typically active at 37.degree. C. and is other than a thermostable
enzyme. In some embodiments, the RNase H enzyme is RNase II.
In some embodiments, the cleavable moiety is a site cleavable by an
apurinic/apyrimidinic (AP) endonuclease when the oligonucleotide or
blocked primer is hybridized to the nucleic acid template forming a
double stranded duplex. An abasic, or baseless, site is cleavable
by AP endonucleases and includes an apurinic site, an apyrimidinic
site or a spacer. In some embodiments, AP endonucleases include
Endonuclease IV (Endo IV), APE 1 or APE 2. See Example 6. In some
embodiments, the endonuclease, including AP endonucleases, is an
enzyme that is active at 37.degree. C. and/or is other than a
thermostable endonuclease.
The oligonucleotide or primer configuration can be any of those
disclosed herein as to length of the 5' domain, length of the 3'
domain and/or location of the cleavable moiety. In some
embodiments, the first oligonucleotide or blocked primer is between
15 and 200, 15 and 150, 15 and 100, or 15 and 50 nucleotides long,
and wherein the cleavable moiety is more than 5, 6, 7, 8, 9. 10,
11, 12, 13, 14, or 15 nucleotides away from the 3' terminus of the
oligonucleotide. In some embodiments, the blocked oligonucleotide
primer (e.g. first or second oligonucleotide; forward or reverse
primer) includes a 5' domain and a 3' domain separated by the one
or more nucleotides that are cleavable by RNase H, wherein the 5'
domain is 10 to 50 nucleotides in length and the 3' domain is 10 to
50 nucleotides in length. In some embodiments, the RNase H enzyme
is RNase HII.
In some embodiments, the first oligonucleotide is between 15 and
200 nucleotides long, and wherein the abasic site or spacer is more
than 5 nucleotides away from the 3' terminus of the
oligonucleotide. In some embodiments, the blocked oligonucleotide
primer includes a 5' domain and a 3' domain separated by an abasic
site or spacer that is cleavable by an AP endonuclease, wherein the
5' domain is 10 to 50 nucleotides in length and the 3' domain is 10
to 50 nucleotides in length. In some embodiments, the AP
endonuclease is Endo IV or APE 1.
In some embodiments, the first oligonucleotide is a universal
primer, wherein the universal primer can include any of the primer
configurations disclosed herein. In some embodiments, the method of
cleaving a hybridized oligonucleotide includes the use of both a
first and second oligonucleotide, e.g. a forward and reverse primer
that bind to complementary strands of the template nucleic acid in
reverse orientation. In some embodiments, the reaction mixture
further includes a second oligonucleotide that binds to the target
nucleic acid on a complementary strand to the first binding site
and in a reverse orientation, and wherein the second
oligonucleotide is not extendable by the polymerase.
In some embodiments, the method of cleaving a double stranded
template nucleic acid is carried out according to the following
steps: forming a reaction mixture comprising the template nucleic
acid and a first oligonucleotide comprising a cleavable moiety,
wherein the first oligonucleotide is not extendable by a
polymerase; exposing the reaction mixture to an endonuclease enzyme
in the presence of reaction conditions permissive for hybridization
between the first oligonucleotide and a first binding site on the
template nucleic acid, wherein at least a portion of the first
oligonucleotide hybridizes to the template; and cleaving the first
oligonucleotide at the cleavable moiety with the endonuclease,
wherein the endonuclease cleaves the oligonucleotide more
efficiently at 37.degree. C. than at 60.degree. C., wherein the
cleavage step occurs at a temperature below 42.degree. C., or a
combination thereof.
In some embodiments, the endonuclease cleaves a significant
fraction of the first oligonucleotide present in the reaction
mixture within 30 or 60 minutes of exposing the hybridized primer
and template nucleic acid to the endonuclease. To determine whether
the endonuclease cleaves a significant fraction of oligonucleotides
present, as a non-limiting example, the oligonucleotides before
cleavage can be non-extendable by a polymerase wherein when the
oligonucleotide is cleaved extension can occur and a product of the
extension can be detected. In some embodiments, the nucleic acid
template is a member of a polynucleotide library and the method is
carried out on a plurality of nucleic acid templates of the
polynucleotide library, wherein each member of the polynucleotide
library comprises the first primer binding sequence. Accordingly,
the method can be combined with amplification methods, such as RPA,
wherein the reaction mixture further comprises recombinase and
polymerase enzymes to amplify the target nucleic acid. The
recombinase and polymerase enzyme can be any of those disclosed
herein, and are typically other than thermostable enzymes. In some
embodiments, the reaction mixture remains below 42.degree. C. for
both the cleavage and amplification. Accordingly, the cleavage and
amplification are carried at a temperature below 42.degree. C.
In some embodiments, the first primer is a universal primer, the
reaction mixture is subject to amplification conditions, and at
least two of the nucleic acid templates are amplified to form
substantially monoclonal populations. The first primer can be
immobilized on a solid support or the amplification method
comprises bridge amplification or emulsion amplification. In some
embodiments, the amplification is carried out for at least 10
cycles using the first oligonucleotide and the second
oligonucleotide. In some embodiments, the cleavage and 10 cycles
are carried out in less than 15, 30, or 60 minutes.
In some embodiments, provided herein are reaction mixture
compositions, wherein the reaction mixture comprises a population
of cleavable primers, wherein the population of cleavable primers
comprises at least 10 primers that bind to at least 10 target
binding sites on a mammalian genome, wherein the cleavable primers
comprise a 5' domain and a 3' domain separated by one or more
nucleotides that are cleavable by an RNase H, wherein the 5' domain
is 10 to 50 nucleotides in length and the 3' domain is 10 to 50
nucleotides in length. In alternative embodiments, the reaction
mixture comprises a population of cleavable primers, wherein the
population of cleavable primers comprises at least 10 primers that
bind to at least 10 target binding sites on a mammalian genome,
wherein the cleavable primers comprise a 5' domain and a 3' domain
separated by an abasic site or spacer that is cleavable by an AP
endonuclease, wherein the 5' domain is 10 to 50 nucleotides in
length and the 3' domain is 10 to 50 nucleotides in length. In some
embodiments, the 3' domain of the cleavable primer is 14 to 25
nucleotides in length.
The reaction mixture compositions can further include a polymerase,
a recombinase or both. Those enzymes can be any of those disclosed
herein and are typically active at 37.degree. C. and/or other than
thermostable enzymes. In some embodiments, the population comprises
at least 100 cleavable primers that are not extendable by a
polymerase or wherein the population comprises at least 1000
cleavable primers that are not extendable by a polymerase.
Those skilled in the art can devise many modifications and other
embodiments within the scope and spirit of the disclosed
inventions. Indeed, variations in the materials, methods, drawings,
experiments examples and embodiments described may be made by
skilled artisans without changing the fundamental aspects of the
disclosed inventions. Any of the disclosed embodiments can be used
in combination with any other disclosed embodiment.
EXAMPLES
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to use the embodiments provided herein, and are
not intended to limit the scope of the disclosure nor are they
intended to represent that the Examples below are all of the
experiments or the only experiments performed. Efforts have been
made to ensure accuracy with respect to numbers used (e.g. amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by volume, and temperature is in degrees Centigrade. It
should be understood that variations in the methods as described
can be made without changing the fundamental aspects that the
Examples are meant to illustrate.
Example 1: Screening of RNase H Cleavable 3' Blocked Primer
Configurations for Use in a Recombinase Polymerase Amplification
Reaction
Experiments were performed that analyzed configurations for blocked
forward primers and blocked reverse primers for use in methods for
performing recombinase polymerase amplification of a nucleic acid
template under isothermal amplification conditions. Configurations
analyzed in these experiments included the length of the 3' domain
of the blocked forward and reverse primers and the identity of the
ribobase, which is the site of RNase H cleavage located between the
5' domain and 3' domain of the 3' blocked primers. See FIG. 2 for a
diagram of the 3' blocked primers and the different configurations
analyzed in these Examples.
Recombinase polymerase amplification (RPA) reactions were performed
using the following protocol in a single reaction vessel with a
single continuous liquid phase in a total reaction volume of
.about.50 .mu.L:
The recombinase source was from a TwistAmp Basic kit (TwistDx,
Cambridge, Great Britain). Dehydrated pellets in the kit contain
uvsX recombinase, uvsY recombinase loading protein, gp32 protein,
Sau DNA polymerase, dNTPs, ATP, phosphocreatine and creatine
kinase. One pellet from a TwistAmp Basic kit was rehydrated in 29.5
uL of Rehydration buffer supplied from the kit in a 0.2 .mu.L PCR
tube. The recombinase solution was vortexed and spun, then
iced.
The template DNA (at various concentrations) in a volume of 0.5
.mu.L was added to the reaction tube, vortexed and then spun. The
template DNA was either a genomic DNA, or a DNA library made with
adapters for amplification.
The 3' blocked primers with a single ribobase were synthesized by
IDT (Coralville, Iowa) in various configurations of long and short
5' and 3' domains designated as V1-V6. See FIG. 2 and FIG. 10. A
long 5' domain is greater than 25 nucleotides in length and a long
3' domain is equal to, or greater than 14 nucleotides in length,
whereas a short 5' domain is 15-25 nucleotides in length, and a
short 3' domain is 4-6 nucleotides in length. The medium length 3'
domain tested was 10 nucleotides in length. In these experiments
the 3' blocking group was a 3'-C3 spacer, but it is understood that
other 3'-blocking groups such as a phosphate, biotin, amine, etc.
can be used with the 3' blocked primer.
Forward and reverse primers (various amount of 10 uM stocks) and
RNase HII (NEB, Ipswich, Mass.; various unit amount) were added to
the reaction tube. Additional low-TE buffer (10 mM Tris-HCl, 0.1 mM
EDTA, pH 8.0) was added to top off the total volume to 42.5 .mu.L.
The recombinase reaction mix was vortexed, spun, and placed on ice.
Next, 7.5 .mu.L of 54 mM Mg-acetate was added to the inside lid
surface of the PCR tube containing the recombinase reaction. The
PCR tube was closed, then vortexed and spun, and incubated in a
total 50 .mu.L volume at 37.degree. C., for 30 min to 50 minutes in
the thermocycler. The reaction was stopped by adding EDTA at 20 mM
final concentration, then purified with PureLink.RTM. PCR
Purification kit (Thermo Fisher Scientific, Waltham, Mass.).
Amplified product was eluted in 50 .mu.L elution buffer supplied in
the kit. A portion of the product was assessed on E-Gel.RTM. EX
Agarose Gels, 2% (Thermo Fisher Scientific, Waltham, Mass.)
containing a nucleic acid stain, such as SYBRGOLD.
Two separate experiments were conducted to determine the optimal 5'
and 3' domain length as well as the choice of ribobase for the RPA
reaction. The product from the above RPA reaction was obtained
using 1 pM of template (100 bp insert library with a tailed-A (57
bp) and P1B (53 bp) adapters with an expected amplicon size of
about 210 bp), 400 nm each of 3' blocked primers and 8 mM of
Mg-acetate, wherein 15 .mu.L of the 50 .mu.L purified RPA reaction
product was loaded into wells of the agarose gels and the DNA
fragments separated by electrophoresis. See FIGS. 3-5 and Tables
1-2.
Table 1 provides the tested primer designs wherein V1 (short 5'
domain and short 3' domain), V2 (long 5' domain and long 3' domain)
and V3 (long 5' domain and short 3' domain) correspond to the 3'
blocked primers of FIG. 2 and the ribobase is cysteine (rC) or
uracil (rU). MM is a mismatched base following the 3' domain. See
FIG. 3 for the gel that corresponds to Table 1.
TABLE-US-00001 TABLE 1 Configurations for screening V1, V2 and V3
primer designs Lane Primer configuration Results M NEB Low MW
ladder N/A 1 Regular short (20mer) primers Significant primer
dimers (nonspecific bands) 2 Regular long (46-53mer) primers
Significant primer dimers (nonspecific bands) 3 3' blocked primers.
V1-rC No detectable results 4 3' blocked primers. V2-rC
Amplification band present with no primer dimers 5 3' blocked
primers. V2-MM-rC Amplification band present with no primer dimers
6 3' blocked primers. V2-rU Amplification band present with no
primer dimers 7 3' blocked primers. V3 No detectable product
band
The results from the use of the V2 primer configuration indicated
the choice of ribobase is not critical and that the presence of a
3' mismatch base is optional. In comparing the primer
configurations of V1, V2 and V3 the results indicated a short (4-6
nucleotides) 3' domain was inefficient in amplifying nucleic acid
under the RPA reaction conditions analyzed in this experiment, with
either a long or short 5' domain. However, surprisingly, a
relatively long (.gtoreq.14 nucleotides) 3' domain with a long 5'
domain resulted in efficient amplification of template DNA.
Accordingly, in some embodiments, the methods for amplifying a
nucleic acid template provided herein, include a 3' blocked primer
that includes a 5' domain and a 3' domain separated by a ribobase,
wherein the 3' domain is .gtoreq.14 nucleotides in length, such as
14 to 30 nucleotides in length.
To further elucidate the configuration of 5' and 3' domain length,
a comparison between V4 (long 5' domain and medium 3' domain) and
V5 (short 5' domain and long 3' domain) was performed. Table 2
provides the tested primers wherein V4 and V5 correspond to the 3'
blocked primers of FIG. 2 and the ribobase is guanine (rG) or
uracil (rU). See FIGS. 4 and 5 for the gels that corresponds to
Table 2; the gel of FIG. 5 was exposed for a longer period of time
to the detection reagent resulting in the detection of a weak
product band in lane 3 (V4-rU).
TABLE-US-00002 TABLE 2 Primer configuration screening for V4 and V5
as compared to V2-rU Lane Primer configuration Results M NEB Low MW
ladder N/A 1 3' blocked primers. V2-rU Amplification band present
with no primer dimers 2 3' blocked primers. V5-rG/rU Amplification
band present with no primer dimers 3 3' blocked primers. V4-rU Weak
(FIG. 5) or no detectable (FIG. 4) amplification product band with
no primer dimers
The results of the comparison between V4 and V5 indicated: 1) due
to the presence of a weak band in FIG. 5 for V4, that there was
some amplification where the 3' domain of the 3' blocked primer had
a length of 10 nucleotides, in embodiments less than 14 nucleotides
but more than 6 nucleotides; and, 2) a long 3' domain (.gtoreq.14
nucleotides) efficiently amplified DNA in the RPA reaction with a
5' domain of 15 nucleotides or greater. Accordingly, in some
embodiments, the methods for amplifying a nucleic acid template
provided herein, include a 3' blocked primer that includes a 5'
domain and a 3' domain separated by a ribobase, wherein the length
of the 3' domain is equal to or greater than 7, 8, 9, 10, 11, 12,
13 or 14 nucleotides on the low end of the range and equal to or
less than 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50
nucleotides on the high end of the range. In embodiments the length
of the 3' domain is from about 7 to 25, about 10 to 25 or about 14
to 25 nucleotides and the length of 5' domain is from about 15 to
60, about 15 to 40 or about 15 to 25 nucleotides.
The results of these experiments indicated efficient amplification
using 3' blocked primers with a 5' DNA domain equal to or greater
than (.gtoreq.) 15 nt and 3' DNA domain equal to or greater than
(.gtoreq.) 14 nt (excluding any possible 3' mismatched
nucleotides). The cleaved 3' domain by RNase H enzyme is greater
than or equal to (.gtoreq.) 15 nucleotides when including the
ribobase in the length. The ribobases uracil, guanine and adenine
are approximately equivalent but the interaction between RNase H
enzyme and cysteine ribobase may result in slower kinetics under
certain reaction conditions. In the experiments described herein,
the primers V2 and V5 produced amplification product with no
detectable nonspecific amplification. Accordingly, in some
embodiments, the methods for amplifying a nucleic acid template
provided herein, include a 3' blocked primer that includes a 5'
domain and a 3' domain separated by a ribobase, wherein the 5'
domain is at least 25 nucleotides in length and the 3' domain is at
least 14 nucleotides in length. In some embodiments, the 5' domain
of the 3' blocked primer is 15-25 nucleotides in length and the 3'
domain is at least 14 nucleotides in length. In some embodiments,
the length of 5' domain is at least 15 nucleotides and the length
of the 3' domain is at least 14 nucleotides. In yet other
embodiments, the methods for amplifying a nucleic acid template
provided herein, include a 3' blocked primer that includes a 5'
domain and a 3' domain separated by a ribobase, wherein the 5'
domain is 15 to 50 nucleotides in length and the 3' domain is 14 to
50 nucleotides in length.
Example 2: Use of RNase H Cleavable 3' Blocked Primer in a
Recombinase Polymerase Amplification Reaction Reduces Nonspecific
Product Formation
Experiments were performed that analyzed the efficiency of the
blocked forward primers and blocked reverse primers for use in
methods for performing recombinase polymerase amplification of a
nucleic acid template under isothermal amplification conditions as
compared to standard (non-blocked) primers and the amplification of
nonspecific DNA such as primer dimers. The results illustrate that
the cleavable 3' blocked primers provided herein reduce or
eliminate primer dimer formation in RPA methods.
First, 3' blocked primers (forward and reverse) demonstrated an
ability to reduce nonspecific product amplification in a control
reaction performed in the absence of template DNA, as compared to
standard (non-blocked) short and long primers. The RPA reaction was
carried out as described in Example 1 except that no template was
included in the reaction mixture, which included 400 nm each of 3'
blocked primers, with or without RNase HII enzyme, and 8 mM of
Mg-acetate, wherein 15 .mu.L of the 50 .mu.L purified RPA reaction
product was loaded into wells of the agarose gel and the DNA
fragments separated by electrophoresis. See FIG. 6 and Table 3.
Non-specific amplification was tested in a no template control
reaction. 3' blocked primers (V2-rU design) were compared to
standard (non-blocked) long and short primers and V2-rU (long 5'
domain and long 3' domain) (Table 3). The results are shown on FIG.
6. Regular (unblocked) primers produced significant amounts of
spurious amplification products in the no template control
reactions. On the other hand, using the V2-rU 3' blocked primers,
only minimal nonspecific products were detected.
TABLE-US-00003 TABLE 3 Reduced non-specific amplification product
with V2-rU as compared to non-blocked primers Lane Primer
configuration Results M NEB Low MW ladder N/A 8 NTC: Standard short
primers Nonspecific amplification product present 9 NTC: Standard
long primers Nonspecific amplification product present 10 NTC: 3'
blocked primers V2-rU Minimal nonspecific amplification product
Next, the formation of nonspecific products such as primer dimers
was analyzed with the use of 3' blocked primers compared to
standard (non-blocked) primers wherein the 3' blocked primers were
designed to have functional primers after cleavage (by RNase H
enzyme) that match and have the same sequence as the standard
primers used in the RPA reaction. Recombinase polymerase
amplification (RPA) was performed using 100 ng of template (E. coli
genomic DNA), 480 nm each of blocked and standard primers (SEQ ID
Nos:2-5), RNase H enzyme and 8 mM of Mg-acetate, wherein 5 or 15
.mu.L of the 50 .mu.L purified RPA reaction product was loaded into
wells of the agarose gel, and the DNA fragments were separated by
electrophoresis. See FIG. 7 and Table 4.
The following standard (non-blocked) primers were used:
TABLE-US-00004 268-Forward primer: (SEQ ID NO: 2) ACA CGG TCC ADA
CTC CTA CGG GAG GCA GCA (e.g., where according to IUPAC "D" is
selected at random from A, T or G). 268-Reverse primer: (SEQ ID NO:
3) GCG GCT GCT GGC ACG GAG TTA GCC GGT GCT
The following 3' blocked primers, wherein the underlined sequence
is identical to the corresponding forward or reverse standard
(non-blocked) primer after cleavage, were used:
TABLE-US-00005 268-Forward primer-rG: (SEQ ID NO: 4) ACA CGG TCC
ADA CTC CTA CGG GAG GCA GCA rGTG GGG AAT ATT GCA C-block
268-Reverse primer-rU: (SEQ ID NO: 5) GCG GCT GCT GGC ACG GAG TTA
GCC GGT GCT rUCT TCT GCG GGT AAC G-block
The following E. coli amplicon 268 was generated from template gDNA
(202 bp) as a result of the RPA reaction:
TABLE-US-00006 (SEQ ID NO: 1) ACA CGG TCC AGA CTC CTA CGG GAG GCA
GCA GTG GGG AAT ATT GCA CAA TGG GCG CAA GCC TGA TGC AGC CAT GCC GCG
TGT ATG AAG AAG GCC TTC GGG TTG TAA AGT ACT TTC AGC GGG GAG GAA GGG
AGT AAA GTT AAT ACC TTT GCT CAT TGA CGT TAC CCG CAG AAG AAG CAC CGG
CTA ACT CCG TGC CAG CAG CCG C
Table 4 provides the tested primers, standard primers and 3'
blocked primers wherein the 5' domain was 30 nucleotides in length
and the 3' domain was 15 nucleotides in length. Each RPA product
was loaded at 5 .mu.l and 15 .mu.l in two wells, respectively. See
FIG. 7 for the gel that corresponds to Table 4.
TABLE-US-00007 TABLE 4 3' Blocked primers eliminate primer dimer
product in gDNA amplification as compared to standard (non-blocked)
primers. Lane Primer & Exp. Configuration Results M NEB low
molecular weight N/A ladder 1 Standard primers; 5 .mu.L Primer
dimer product and loaded amplification product present 2 3' blocked
primers; with No detectable primer dimer RNaseH enzyme; 5 .mu.L
product; amplification product loaded present 3 3' blocked primers;
No No detectable primer dimer RNaseH enzyme control; product or
amplification product 5 .mu.L loaded 4 Standard primers; 15 .mu.L
Primer dimer product and loaded amplification product present 5 3'
blocked primers; with No detectable primer dimer RNaseH enzyme; 15
.mu.L product; amplification product loaded present 6 3' blocked
primers; No No detectable primer dimer RNaseH enzyme control;
product or amplification product 15 .mu.L loaded
As shown in FIG. 7, amplification using standard (non-blocked) RPA
primers under these conditions, generated detectable primer dimers.
On the other hand, primer dimers were not detectable after
amplification using the 3' blocked primers of the invention, having
a 5' domain after cleavage, that contains the same sequence as the
standard primers. Furthermore, primer dimers were not detected when
the 3' blocked primers were uncleaved (no RNase H enzyme), and
therefore fail to produce detectable amplification product.
Accordingly, this experiment demonstrates that the blocked forward
primers and blocked reverse primers when used in methods for
performing recombinase polymerase amplification of a nucleic acid
template under isothermal amplification conditions efficiently
amplify template DNA without substantially generating nonspecific
product such as primer dimer product.
Example 3: Titration of RNase H Enzyme for Use in a Recombinase
Polymerase Amplification Reaction with RNase H Cleavable 3' Blocked
Primers
Experiments were performed that analyzed various amounts of RNase H
enzyme, including a "prohibiting" amount, a "limiting" amount and
an "excess" amount for use in the methods for performing
recombinase polymerase amplification of a nucleic acid template
under isothermal amplification conditions with the blocked forward
primers and blocked reverse primers. Not to be limited by theory,
in the initial stage of amplification where low copies of template
DNA is present, primer-template duplex "D-loop" is considered a
rare event. A certain amount of RNase H enzyme is required to
cleave the duplex to activate the primer before the D-loop
dissociates. If RNase H enzyme is too low, the chance of an RNase
enzyme interacting with a blocked primer bound to a template may
become mathematically impossible so that amplification does not
progress. In that instance, the RNase H enzyme is considered to be
a "prohibiting" amount. When more RNase H enzyme is present,
cleavage of the template-bound blocked primer becomes possible and
more efficient, while still not fully efficient. In that instance,
amplification progresses at a reduced rate, although such an
amplification reaction can still achieve sufficient amplification
product if allowed a longer reaction time. In that instance, the
RNase H enzyme is considered a "limiting" amount. An "excess"
amount of the RNase H enzyme ensures the RPA reaction proceeds
based on the kinetics of the polymerase and other components in the
reaction mixture and not the enzyme needed to remove the blocking
group, which would otherwise be a rate limiting step for primer
extension. In that instance, RNase H enzyme is present in an amount
to guarantee full cleavage of the ribobase-containing primer, and
is "non-limiting"; RNase H enzyme will not slow down or diminish
amplification as compared to a "prohibiting" or "limiting" amount
of enzyme. In some embodiments, the RNase H enzyme is present in
the RPA reaction mixture in an excess amount. The titration
experiments described herein empirically determine an excess amount
of RNase HII enzyme with the V2 and V5 3' blocked primer
configurations of the invention in the RPA methods for amplifying
DNA template.
The RPA experiment was conducted following the methods of Example 1
using template library DNA (100 bp insert library with a tailed-A
(57 bp) and P1B (53 bp) adapters with an expected amplicon size of
about 210 bp) and 400 nm each of 3' blocked primers (V2-rU). The
amplification reaction was performed at 37.degree. C. for 50
minutes. The RNase HII was concentrated 20.times. from the original
5 U/uL concentration. 100 U in a 1 uL volume (20.times.
concentrate) was tested as compared to 5-20 U in a 1-4 uL volume
(standard concentration from NEB).
FIG. 8A provides the identity of samples loaded onto gels the
tested RNase H enzyme and the 3' blocked primers (V2-rU) wherein
the 5' domain is 25 nucleotides in length and the 3' domain is 14
nucleotides in length. Each RPA product was loaded at 15 .mu.l in a
wells of the gel. As shown in FIG. 8A, when amplified for 50
minutes, as low as 5 U of RNase HII produced detectable product
with exemplified V2-rU 3' blocked primer configuration. All
concentrations of RNase HII enzyme used produced detectable
product, with increasing amounts of RNase HII enzyme yielding more
product, as seen with brighter product bands, however the RNase H
enzyme is saturated at 20 U, i.e. an excess amount. RNase H enzyme
above 20 U, including up to 200 U had no detrimental effect on the
product produced. In the control lane with 3' blocked primers and
no RNase HII enzyme, no product was detected. Of note, in the
control lane with standard non-blocked primers (and no RNase HII)
non-specific product bands were detected confirming results
observed in Example 2.
In this experiment, based on the intensity of the product bands,
5-10 U of RNase HII enzyme is considered limiting for the V2 primer
configuration in a 50 minute reaction while 20 U-200 U is
considered in excess.
Titration of RNase H enzyme was further analyzed with primer
configurations V2-rU, V2-rC and V5. FIG. 8B provides the identity
of samples loaded onto gels, the tested RNase H enzyme and the type
of 3' blocked primers. Each RPA product was loaded at 10 .mu.l in
wells of the gel. As shown in FIG. 8B, when amplified for 50
minutes, as low as 5 U of RNase HII produced detectable product
with exemplified V2-rU and V2-rC 3' blocked primer configuration
(lanes 3 and 6, respectively) but is "prohibiting" at 2.5 U wherein
no detectable product band was generated (lanes 2, 5 and 8). In
this experiment, 5-10 U of RNase HII enzyme is considered
"limiting" for the V2 primer configuration in a 50 minute reaction
based on the intensity of the product bands.
While relatively less efficient under other limiting reaction
conditions (e.g. shorter reaction time), ribobase rC (lanes 5-7)
performed similarly to rU primers (lanes 2-4) in V2 configurations
in amount of product generated. Accordingly, in some embodiments
the ribobase in the 3' blocked primers can be rC. The V5 primer
with short 5' domain (lanes 8-9) is less efficient as compared to
V2, and may require more RNase HII to achieve a similar level of
amplification under similar reaction condition. In this experiment,
2.5-5 U of RNase H enzyme is considered "prohibiting" and 10 U is
considered "limiting" for V5 primer configuration in a 50 minute
reaction based on the intensity of the product bands.
For the control (lane 1) of non-blocked standard primers with no
RNase HII enzyme, significant non-specific product bands were
observed.
RNase H enzyme was further analyzed at different amplification
reaction temperatures to determine an optimal temperature range for
RNase HII enzyme.
The RPA experiment was conducted following the methods of Example 1
using 400 nm each of 3' blocked primers (V2-rU) and 10 U in a 2
.mu.L volume of RNase HII from NEB, wherein the reaction was
incubated from a range of 37.degree. C. to 42.degree. C. for 50
minutes. Each RPA product was loaded at 5 .mu.l in a wells of the
gel. FIG. 8C provides the identity of samples loaded onto gels, the
tested RNase H enzyme, the 3' blocked primers and reaction
temperature. As shown in FIG. 8C, RNase HII performs optimally at
37.degree. C., with a faint band present in lane 3 (40.degree. C.
reaction temperature) and no product band visible in lane 4
(42.degree. C. reaction temperature). As will be appreciated,
different RNase H enzymes may perform optimally at different
temperatures and such conditions may be empirically determined with
the 3' blocked primers in the RPA amplification methods.
Example 4: Use of Alternative Reaction Mixture in Recombinase
Polymerase Amplification Reaction with RNase H Cleavable 3' Blocked
Primers
RPA experiments were performed that analyzed an exemplary
alternative base RPA reaction mixture formulation as compared to
the base reaction mixture in the Examples above, which were formed
by hydrating a commercially available pellet (TwistDx, Cambridge,
Great Britain), adding blocked forward and reverse primers, RNase
HII, additional low-TE buffer, and Mg-acetate.
Reaction mixtures prepared using commercially available pellets
from TwistDx contain uvsX recombinase, uvsY recombinase loading
protein, gp32 protein, Sau DNA polymerase, dNTPs, ATP,
phosphocreatine and creatine kinase. An exemplary alternative
reaction mixture was prepared in pellet form, that included the
same components as the commercially available reaction mixture
above except that Sau polymerase was replaced with a mixture of Sau
polymerase and a T7 DNA polymerase, with thioredoxin. After the
reaction mixture pellet was hydrated, RNase H enzyme, and 3'
blocked primers were added. Except for the reaction mixture
modifications noted, the RPA reaction was performed as described in
Example 1 using the 3' blocked primer configurations of V1, V2-rU,
V2-rC, V2-MM-rC, and V3-rC. Standard (non-blocked) primers were
used as a control.
The RPA reaction was performed following the protocol in Example 1
using 1 pM of template DNA (100 bp insert library with a tailed-A
(57 bp) and P1B (53 bp) adapters with an expected amplicon size of
about 210 bp), 400 nm each of primers, 45 U or 20 U RNase HII
enzyme (with 3' blocked primers) and 8 mM of Mg-acetate. After the
reaction was performed and stopped, 15 uL of the 50 uL purified RPA
reaction product was loaded into wells of the agarose gel and the
DNA fragments were separated by electrophoresis.
Table 6 provides the identity of those samples loaded onto the gel.
As seen in FIG. 9 and summarized in Table 6, amplification products
of the expected size as well as primer dimers, were seen in
amplifications using standard primers (lane 1). For the 3' blocked
primers, results were similar exemplary alternative reaction
mixture to those obtained using the standard commercial reaction
mixture. No reaction products were detectable in samples generated
using V1 or V3 primers, which have the relatively short 3' domain,
or samples with 20 U of RNase HII or a no RNase control. All other
samples that were generated using 3' blocked primers contained the
expected amplification product in detectable quantities.
TABLE-US-00008 TABLE 6 Pellet formulation is compatible and results
in efficient DNA template amplification with the forward and
reverse 3' blocked primers. Lane Primer & Exp. Configuration
Results M NEB Low MW ladder N/A 1 Regular primers; TwistDx pellet
Detectable amplification control product and nonspecific
amplification product 2 3' blocked primers, V2-rU; TwistDx
Detectable amplification pellet control; 45 U RNase H product 3
Regular primers; alternative exemplary Detectable amplification RPA
reaction mixture formulation; product 45 U RNase H 4 3' blocked
primers, V1; alternative No detectable exemplary RPA reaction
mixture amplification product formulation; 45 U RNase H 5 3'
blocked primers, V2-rC; alternative Detectable amplification
exemplary RPA reaction mixture product formulation; 45 U RNase H 6
3' blocked primers, V2-MM-rC; Detectable amplification alternative
exemplary RPA reaction product mixture formulation; 45 U RNase H 7
3' blocked primers, V3-rC; alternative No detectable exemplary RPA
reaction mixture amplification product formulation; 45 U RNase H 8
3' blocked primers, V2-rU; alternative Detectable amplification
exemplary RPA reaction mixture product formulation; 45 U RNase H 9
3' blocked primers, V2-rU; alternative No detectable exemplary RPA
reaction mixture amplification product formulation; 20 U RNaseH2 10
3' blocked primers+, V2-rU; alternative No detectable exemplary RPA
reaction mixture amplification product formulation; No RNase H
(control)
In summary, the results confirmed that V1 and V3 primer
configurations, which have a relatively short 3' domain (4-6
nucleotides) length are inefficient in amplifying template DNA, but
that V2 primers with either the commercially available pellet and
reaction mixture formulation or the alternative exemplary RPA
pellet and reaction mixture formulation efficiently amplified the
template DNA. Furthermore, the experiment illustrates the
robustness of the 3' blocked primers to different base reaction
mixture pellet and soluble reaction mixture formulations.
Accordingly, In some embodiments, provided herein, is a reaction
amplification method, such as RPA, performed using a dehydrated
pellet formulation (which is rehydrated prior to use).
Example 5: Amplification of Template with RNase H Cleavable 3'
Blocked Primers Attached to a Solid Support
This example illustrates clonal amplification of a DNA template
using a recombinase polymerase amplification reaction wherein at
least one of the primers of a primer pair is a 3' blocked primer,
wherein the 3' blocked primer is attached to a support. See FIG. 11
for exemplified primers of the invention, wherein the blocking
group C3 spacer is represented as 3spC3 in the primer sequences.
The 3spC3 spacer can include phosphoramidite (available from
Integrated DNA Technologies, Coralville, Iowa).
As a non-limiting example, the RPA method can be performed
essentially using the methods described in Example 1 in a single
reaction vessel in a single continuous liquid phase, wherein a
reverse 3' blocked primer with a V2 or V5 configuration is attached
to a bead and the forward primer (3' blocked primer of the
invention or standard non-blocked primer) is added in solution to
the reaction mixture. Total reaction volume is 50 .mu.L to 1.2 mL.
The following discusses a reaction in 300 .mu.L volume.
Beads, such as 1.25 .mu.L from an 80 million/.mu.L stock (100
million beads) with the attached reverse primer are added in a 1.5
mL tube (tube 1). The required bead count can vary depending on the
sequencing chip (provided the bead-primer-template-MgOAc mixture
volume does not exceed 40% of the reaction volume) but is typically
in the range from 20 million to 1 billion beads for a reaction
scale of 50 .mu.L up to 2.4 mL. For example, the bead count used
can be from 10 to 100 million/.mu.L. A forward primer, standard or
3' blocked primer of the invention, (1.2 .mu.L of a 100 .mu.M
stock) is added to the bead tube (tube 1), followed by vortexing
and spinning to a final concentration of 0.4 .mu.M in the final
reaction. The immobilized blocked reverse primer sequence can be
one of the following:
TABLE-US-00009 (SEQ ID NO: 6) 5'- CCT ATC CCC TGT GTG CCT TGG CAG
TCT CAG CCrU CTC TAT GGG CAG TCG A/3SpC3/- 3'; (SEQ ID NO: 7)
5'-CCA CTA CGC CTC CGC TTrU CCT CTC TAT GGG CAG /3SpC3/; or (SEQ ID
NO: 8) 5'-C CTC CGC TTT CCT CTC TrAT GGG CAG TCG GTG AT
/3SpC3/.
The forward primer of a standard (non-blocked) configuration, or a
3' blocked primer with a V5 configuration, is added to the bead
tube (tube 1), followed by vortexing and spinning. The solution
forward primer sequence can be one of the following:
TABLE-US-00010 (SEQ ID NO: 9) 5'- CCA TCT CAT CCC TGC GTG TC -3';
(SEQ ID NO: 10) 5'-CCA TCT CAT CCC TGC GTG TCT CCG AC-3'; (SEQ ID
NO: 11) 5'-CCA TCT CAT CCC TGC rGTG TCT CCG ACT CAG /3SpC3/; or
(SEQ ID NO: 12) AAC GAT CCA TCT CAT CCC TGC rGTG TCT CCG ACT CAG
/3SpC3/
A biotinylated forward primer (0.12 uL of a 10 .mu.M stock) is
added to the bead tube (tube 1), followed by vortexing and
spinning. The biotinylated forward primer sequence can be:
5'Bio-CCA TCT CAT CCC TGC GTG TC-3' (SEQ ID NO:13).
Various volumes of polynucleotide library (at 100 pM concentration)
is added to the bead tube (tube 1), followed by vortexing and
spinning. The library volume can be varied depending on the desired
DNA-to-bead ratio of 3:1, 2:1, 1:1, 1:1.7, 1:3, 1:5, 1:10. For
example, the DNA-to-bead ratio can be 1:1 to 1:1.5.
The rehydrated recombinase mix (tube 2, reconstituted in 180 .mu.L
rehydration buffer to a volume of approximately 185 .mu.L) is added
to the bead tube (tube 1), followed by vortexing and spinning.
Various amount of RNase H (at 20.times. concentrate from original
NEB product) is added to the reaction tube, followed by vortexing
and spinning. The volume is filled to 225 .mu.L with low-TE.
75 .mu.L of iced 28 mM Mg-acetate in sieving agent is added to the
bead tube, followed by vortexing and spinning, and put back on ice
for 10 seconds, and incubated at 37.degree. C. for 30-60 minutes on
the heat block.
The reaction is stopped by adding 50 .mu.L 500 mM EDTA, and 100
.mu.L 1% SDS. The reaction tube is then topped off to 1 mL with TE
buffer, followed by vortexing and spinning.
The beads are enriched by binding the biotinylated polynucleotides
with paramagnetic beads conjugated with streptavidin (MyOne.TM.
Bead from Dynabeads).
The enriched beads are loaded into an ION TORRENT ion-sensitive
chip and a standard sequencing reaction is conducted.
The experiment disclosed above in this Example was performed using
SEQ ID NO:6 attached to beads as the universal reverse 3' blocked
primer, SEQ ID NO:9 as the universal non-blocked standard primer in
solution and SEQ ID NO:13 as the biotinylated forward primer for
bead enrichment. Barcoded template DNA libraries (100 bp insert
library with a tailed-A (57 bp) and P1B (53 bp) adapters with an
expected amplicon size of about 210 bp) (See FIG. 10) were used.
Each of the barcoded libraries was amplified in individual
reactions, for a total of four samples, using 2 to 4 uL of
20.times. RNase H enzyme, See FIG. 12A.
Following amplification, the beads from each barcoded library were
combined and sequenced on the same ION TORRENT sequencing chip. The
amplification of template was successful on beads immobilized with
a 3' blocked primer from a reaction in a single continuous liquid
phase. The RPA amplification methods using an immobilized 3'
blocked primer of the invention and RNase H enzyme generated
monoclonally amplified templates on beads from a template library,
which were subsequently sequenced. The RNase H enzyme, which may be
rate limiting for the amplification step, was analyzed by using two
different volumes (2 .mu.L and 4 .mu.L) of the 20.times. enzyme
(See FIG. 12A) for the impact downstream on the sequencing
reaction. The estimated unit concentration of the 20.times.
concentrate is about 100 U/.mu.l; 2 .mu.L of RNase H contains about
200 U of enzyme and 4 .mu.L contains about 400 U of enzyme. The
results indicated that more enzyme, as compared to half as much,
resulted in better amplification of the template on the beads. See
FIG. 12B, wherein the samples (2 and 4) with about 400 U of enzyme
resulted in more "reads", i.e. more amplified template, as compared
to samples 1 and 3 with half as much (200 U) RNase H enzyme.
Moreover, virtually complete amplification was reached within 40
minutes. Little further gain was achieved with a longer 60 minute
amplification reaction. See FIGS. 12A to 12C.
Example 6: Amplification of Template with an Abasic (Baseless)
Cleavable Blocked Primer in a RPA Amplification Reaction
This example illustrates clonal amplification of a DNA template
using a recombinase polymerase amplification reaction wherein at
least one of the primers of a primer pair is a 3' blocked primer,
wherein the blocked primer contains an abasic cleavable moiety that
is apurinic, apyrimidinic or a spacer. That cleavable moiety is
hydrolyzed by an apurinic/apyrimidinic (AP) endonuclease when the
blocked primer is hybridized to the DNA template forming a double
stranded duplex. Accordingly, the blocked primers comprise a 5'
domain, an abasic cleavable moiety (e.g. baseless), a 3' domain and
a blocking moiety (i.e. the primer is not extendable by a
polymerase).
Class I and II AP endonucleases create a nick in the phosphodiester
backbone at the 5' side of the abasic site leaving a polymerase
extendable 3'-OH on the remaining 5' domain of the primer for
extension. Examples of AP endonucleases include endonuclease IV
(commercially available from Thermo Fisher, Carlsbad, Calif.), APE
1 (commercially available from Thermo Fisher) and APE 2.
The blocked primers with a cleavable abasic site can be synthesized
with an internal abasic furan (e.g., tetrahydrofuran) or spacer
that replaces a nucleotide at the desired cleavage site.
Alternatively, the primers containing an abasic site can be
synthesized with a uracil base replacing a nucleotide at the
desired cleavage site. Prior to use in the methods, that primer can
be treated with Uracil-DNA Glycosylase (UDG) to convert the Uracil
to an abasic site; UDG removes uracil residues from the sugar
moiety of single- and double-stranded DNA without destroying the
phosphodiester backbone.
As a non-limiting example, the RPA method can be performed
essentially using the methods described in Example 1 in a single
reaction vessel in a single continuous liquid phase, wherein a
blocked (non-extendable) primer containing an abasic cleavable
moiety and a biotin blocking moiety with a V2 configuration is
added in solution to the reaction mixture, wherein Endo IV or APE 1
are used as endonucleases replacing RNase H in Example 1.
Amplification using the abasic cleavable blocked (non-extendable)
primers with Endo IV or APE 1 was compared to amplification using
ribobase cleavable blocked (non-extendable) primers of the
invention with a V2 or V3 configuration and RNase H enzyme.
The abasic containing primers were synthesized with a uracil
between the 5' domain and the 3' domain of the blocked primers. The
blocked forward and reverse primer sequence were as follows,
wherein the uracil is converted to an abasic site prior to use:
TABLE-US-00011 Forward AP-15D: (SEQ ID NO: 28) GAA TCT GTC CAT AAG
GTC AGT AAC GAT CCA UCT CAT CCC TGC GTG T-3'biotin Reverse AP-15D:
(SEQ ID NO. 29) CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG CCU CTC TAT
GGG CAG TCG-3'biotin
The ribobase containing blocked primes used in a control RPA
reaction with RNase HII enzyme were selected from the primer pairs
of SEQ ID NO. 18 and SEQ ID NO. 19; SEQ ID NO. 20 and SEQ ID NO.
21; and SEQ ID NO. 23 and SEQ ID NO. 24. See FIG. 10.
Prior to use in the RPA methods, the internal uracil of SEQ ID NO.
28 and 29 was converted to an abasic site by treatment with UDG.
0.2 uL of each uracil-containing blocked primer (100 uM stock), 0.4
uL UDG (1 U/uL, Thermo Fisher Scientific), and 1.2 uL RPA
rehydration buffer (same formulation as described for RPA
reaction), were combined for a total of 2 uL mixture. The mixture
was incubated at 37.degree. C. in a thermocycler for 15 min for
conversion to AP-containing primers. The 2 uL treated primer
mixture was then added to RPA reaction (50 uL reaction) as the
primer mix, so that each primer was 400 nM in the RPA reaction. The
primer mix volume can be adjusted for other desired primer
concentrations.
Hence, the RPA method was performed using SEQ ID NO. 28 and 29,
containing an abasic site at the internal uracil, and the AP
endonuclease endo IV (sourced from NEB and Thermo Fisher) or APE 1
(NEB). Template DNA libraries with an expected amplicon size of
about 123bp were used starting with 1 pM of template. Each of the
libraries was amplified in individual reactions, for a total of
four samples, using 4 .mu.L of commercial concentrations of Endo IV
from NEB and Thermo Fisher and 2 .mu.L or 5 .mu.L of 10.times. Endo
IV (Thermo Fisher). See FIG. 13. The RPA reaction mixture was
incubated for 17 hours at 37.degree. C.
The amplification of template was successful with a blocked primer
containing an abasic site from a reaction in a single continuous
liquid phase when using 10.times. Endo IV. However, nonspecific
products were also present, along with the template. The RPA
amplification method using a primer with an abasic residue and
endonuclease IV cleavage, could be optimized to reduce or eliminate
the nonspecific products. Amplification of template was not
successful when using Endo IV at the concentration provided by the
commercial vendor. Not to be limited by theory, the results
indicate the Endo IV enzyme, similar to RNase H, may be rate
limiting for the amplification step, wherein more enzyme, i.e.,
10.times., resulted in better amplification of the template. In
some embodiments, Endo IV is a viable endonuclease when paired with
abasic 3' blocked primers for use in the RPA methods for
amplification of template DNA.
The RPA method was also performed using SEQ ID NO. 28 and 29,
containing an abasic site at the internal uracil, and the AP
endonuclease APE 1 (NEB) as compared to use of ribobase blocked
primers (SEQ ID NO. 18 and 19) and RNase H enzyme (NEB). Template
DNA libraries with an expected amplicon size of about 123 bp were
used starting with 1 pM of template. Each of the libraries was
amplified in individual reactions, for a total of 10 samples, using
40 U of APE 1, 20 U or 45 U of RNase HII; 100 nm to 800 nm each of
SEQ ID NO 18 and 19 for the RNase H containing reaction mixture and
100 nm or 400 nm of SEQ ID NO. 28 and 29 for the APE 1 containing
reaction mixture. See FIG. 14. The RPA reaction mixture was
incubated for 30 minutes at 37.degree. C.
Amplification of the template was successful for all ten
amplification reactions including the reactions with the abasic
cleavable blocked primers and APE 1 enzyme. No non-specific
amplification product was observed, including primer dimers. See
FIG. 14. In some embodiments, APE 1 is a viable endonuclease when
paired with abasic blocked primers of the invention for use in the
RPA methods for amplification of template DNA.
A third experiment was conducted using RNase HII enzyme as a
control with the primer pairs SEQ ID NO. 18 and 19; SEQ ID NO. 20
and 21 and SEQ ID NO. 22 and 23 (See FIG. 10) as compared with the
use of Endo IV or APE 1 enzyme and primer pair SEQ ID NO. 28 and
29, with and without the internal uracil converted to a cleavable
abasic moiety. The product from the above RPA reaction was obtained
using 1 pM of template, as above, 100 nm each of blocked primers,
20 U of RNase HII, 200 U or 50 U of Endo IV and 40 U of APE 1,
wherein a portion of the 50 .mu.L purified RPA reaction product was
loaded into wells of the agarose gels and the DNA fragments
separated by electrophoresis. See FIG. 15. The RPA reaction mixture
was incubated for 2 hours at 37.degree. C.
The results indicate both Endo IV (when used at a 200 U
concentration) and APE 1 are viable endonucleases, when paired with
abasic blocked primers of the invention, for the amplification of
DNA template in a RPA amplification reaction. Non-specific product
was observed with the use of Endo IV, see FIG. 15 at lane 6 of gel,
but exonuclease activities of the enzyme or contamination of the
commercial enzyme may be the source of the non-specific
amplification. Accordingly, In some embodiments, provided herein,
is a reaction amplification method, such as RPA, performed using
abasic cleavable blocked primers and the endonuclease APE 1 or Endo
IV for cleavage at the abasic site of the blocked primer. Following
cleavage of the primer at the abasic site by either Endo IV or APE
1, the 3' end of the 5' domain is extended and amplification of the
template results as disclosed herein for an RPA amplification
reaction.
SEQUENCE LISTINGS
1
291202DNAArtificial SequenceSynthetic DNA 1acacggtcca gactcctacg
ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc 60tgatgcagcc atgccgcgtg
tatgaagaag gccttcgggt tgtaaagtac tttcagcggg 120gaggaaggga
gtaaagttaa tacctttgct cattgacgtt acccgcagaa gaagcaccgg
180ctaactccgt gccagcagcc gc 202230DNAArtificial SequenceSynthetic
DNA 2acacggtcca dactcctacg ggaggcagca 30330DNAArtificial
SequenceSynthetic DNA 3gcggctgctg gcacggagtt agccggtgct
30447DNAArtificial SequenceSynthetic DNA 4acacggtcca dactcctacg
ggaggcagca rgtggggaat attgcac 47547DNAArtificial SequenceSynthetic
DNA 5gcggctgctg gcacggagtt agccggtgct ructtctgcg ggtaacg
47650DNAArtificial SequenceSynthetic DNA 6cctatcccct gtgtgccttg
gcagtctcag ccructctat gggcagtcga 50734DNAArtificial
SequenceSynthetic DNA 7ccactacgcc tccgcttruc ctctctatgg gcag
34834DNAArtificial SequenceSynthetic DNA 8cctccgcttt cctctctrat
gggcagtcgg tgat 34920DNAArtificial SequenceSynthetic DNA
9ccatctcatc cctgcgtgtc 201026DNAArtificial SequenceSynthetic DNA
10ccatctcatc cctgcgtgtc tccgac 261131DNAArtificial
SequenceSynthetic DNA 11ccatctcatc cctgcrgtgt ctccgactca g
311237DNAArtificial SequenceSynthetic DNA 12aacgatccat ctcatccctg
crgtgtctcc gactcag 371320DNAArtificial SequenceSynthetic DNA
13ccatctcatc cctgcgtgtc 201457DNAArtificial SequenceSynthetic DNA
14gaatctgtcc ataaggtcag taacgatcca tctcatccct gcgtgtctcc gactcag
571553DNAArtificial SequenceSynthetic DNA 15cctatcccct gtgtgccttg
gcagtctcag cctctctatg ggcagtcggt gat 531631DNAArtificial
SequenceSynthetic DNA 16ccatctcatc cctgcgtgtc tccgractca c
311731DNAArtificial SequenceSynthetic DNA 17cctatcccct gtgtgccttg
gcargtctca c 311849DNAArtificial SequenceSynthetic DNA 18gaatctgtcc
ataaggtcag taacgatcca trctcatccc tgcgtgtca 491954DNAArtificial
SequenceSynthetic DNA 19cctatcccct gtgtgccttg gcagtctcag cctctrctat
gggcagtcgg tgaa 542048DNAArtificial SequenceSynthetic DNA
20gaatctgtcc ataaggtcag taacgatcca trctcatccc tgcgtgtc
482152DNAArtificial SequenceSynthetic DNA 21cctatcccct gtgtgccttg
gcagtctcag cctctrctat gggcagtcgg tg 522247DNAArtificial
SequenceSynthetic DNA 22gaatctgtcc ataaggtcag taacgatcca ructcatccc
tgcgtgt 472338DNAArtificial SequenceSynthetic DNA 23gaatctgtcc
ataaggtcag taacgatcca trctcatc 382442DNAArtificial
SequenceSynthetic DNA 24cctatcccct gtgtgccttg gcagtctcag cctctrctat
gg 422542DNAArtificial SequenceSynthetic DNA 25gaatctgtcc
ataaggtcag taacgatcca ructcatccc tg 422632DNAArtificial
SequenceSynthetic DNA 26gaatctgtcc ataagrgtca gtaacgatcc at
322734DNAArtificial SequenceSynthetic DNA 27cctatcccct gtgtgccrut
ggcagtctca gcct 342846DNAArtificial SequenceSynthetic DNA
28gaatctgtcc ataaggtcag taacgatcca uctcatccct gcgtgt
462948DNAArtificial SequenceSynthetic DNA 29cctatcccct gtgtgccttg
gcagtctcag ccuctctatg ggcagtcg 48
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References